Polypeptides Having Lipase Activity and Polynucleotides Encoding Same

ABSTRACT

The present invention relates to isolated polypeptides having lipase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to polypeptides having lipase activity andpolynucleotides encoding the polypeptides. The invention also relates tonucleic acid constructs, vectors, and host cells comprising thepolynucleotides as well as methods of producing and using thepolypeptides.

2. Description of the Related Art

The present invention provides polypeptides having lipase activity andpolynucleotides encoding the polypeptides. The lipase disclosed inGlogauer et al. Microbial Cell Factories 2011, 10:54 shows an amino acidsequence identity of 91.8% with SEQ ID NO: 2.

SUMMARY OF THE INVENTION

The present invention relates to isolated polypeptides having lipaseactivity selected from the group consisting of:

-   -   (a) a polypeptide having at least 92%, at least 93%, at least        94%, at least 95%, at least 96%, at least 97%, at least 98%, at        least 99%, or 100% sequence identity to the mature polypeptide        of SEQ ID NO: 2;    -   (b) a polypeptide encoded by a polynucleotide that hybridizes        under low stringency conditions, medium stringency conditions,        medium-high stringency conditions, high stringency conditions,        or very high stringency conditions with (i) the mature        polypeptide coding sequence of SEQ ID NO: 1, or (ii) the        full-length complement of (i);    -   (c) a polypeptide encoded by a polynucleotide having at least        96%, at least 97%, at least 98%, at least 99%, or 100% sequence        identity to the mature polypeptide coding sequence of SEQ ID NO:        1;    -   (d) a variant of the polypeptide of (a), (b) or (c) comprising a        substitution, deletion, and/or insertion at one or more        positions; and    -   (e) a fragment of the polypeptide of (a), (b), (c), or (d) that        has lipase activity.

The present invention also relates to isolated polynucleotides encodingthe polypeptides of the present invention; nucleic acid constructs;recombinant expression vectors; recombinant host cells comprising thepolynucleotides; and methods of producing the polypeptides.

The present invention also relates to compositions, methods of producinga composition, methods of cleaning a surface, and methods of hydrolyzinga lipid.

DEFINITIONS

Prior to describing the present compositions and methods in detail, thefollowing terms are defined for clarity. Terms and abbreviations notdefined should be accorded their ordinary meaning as used in the art.

Lipase: The terms “lipase”, “lipase enzyme”, “lipolytic enzyme”, “lipidesterase”, “lipolytic polypeptide”, and “lipolytic protein” refers to anenzyme in class EC3.1,1 as defined by Enzyme Nomenclature. It may havelipase activity that may include but are not limited to triacylglycerollipase activity (EC3.1.1.3), monoglyceride lipase activity (EC3.1.1.23),phospholipase activity (several ECs), lysophospholipase activity,ferulic acid esterase activity, cutinase activity (EC3.1.1.74), sterolesterase activity (EC3.1.1.13) and/or wax-ester hydrolase activity(EC3.1.1.50). For purposes of the present invention, lipase activity isdetermined according to the procedure described in the Examples. In oneaspect, the variants of the present invention have at least 20%, e.g.,at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or100% of the lipase activity of the mature polypeptide of SEQ ID NO: 2.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Catalytic domain: The term “catalytic domain” means the region of anenzyme containing the catalytic machinery of the enzyme.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG, or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding a maturepolypeptide of the present invention. Each control sequence may benative (i.e., from the same gene) or foreign (i.e., from a differentgene) to the polynucleotide encoding the polypeptide or native orforeign to each other. Such control sequences include, but are notlimited to, a leader, polyadenylation sequence, propeptide sequence,promoter, signal peptide sequence, and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe polynucleotide encoding a polypeptide.

Expression: The term “expression” includes any step involved in theproduction of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to control sequences that provide forits expression.

Fragment: The term “fragment” means a polypeptide having one or more(e.g., several) amino acids absent from the amino and/or carboxylterminus of a mature polypeptide; wherein the fragment has lipaseactivity. In one aspect, a fragment contains at least 50%, at least 55%,at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, or at least 95% of the number of amino acids ofthe mature polypeptide.

High stringency conditions: The term “high stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at65° C.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Isolated: The term “isolated” means a substance in a form or environmentthat does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., multiple copiesof a gene encoding the substance; use of a stronger promoter than thepromoter naturally associated with the gene encoding the substance). Anisolated substance may be present in a fermentation broth sample.

Low stringency conditions: The term “low stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 25% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at50° C.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 1 to 292 of SEQ ID NO: 2.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving lipase activity. In one aspect, the mature polypeptide codingsequence is nucleotides 30 to 905 of SEQ ID NO: 1.

Medium stringency conditions: The term “medium stringency conditions”means for probes of at least 100 nucleotides in length, prehybridizationand hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mlsheared and denatured salmon sperm DNA, and 35% formamide, followingstandard Southern blotting procedures for 12 to 24 hours. The carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS at 55° C.

Medium-high stringency conditions: The term “medium-high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and either 35%formamide, following standard Southern blotting procedures for 12 to 24hours. The carrier material is finally washed three times each for 15minutes using 2×SSC, 0.2% SDS at 60° C.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the -nobrief option) is usedas the percent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the -nobrief option) is used as the percentidentity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides absent from the 5′ and/or 3′ end of amature polypeptide coding sequence; wherein the subsequence encodes afragment having lipase activity. In one aspect, a fragment contains atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95% ofthe number of nucleotides encoding the mature polypeptide.

Variant: The term “variant” means a polypeptide having lipase activitycomprising an alteration, i.e., a substitution, insertion, and/ordeletion, at one or more (e.g., several) positions. A substitution meansreplacement of the amino acid occupying a position with a differentamino acid; a deletion means removal of the amino acid occupying aposition; and an insertion means adding an amino acid adjacent to andimmediately following the amino acid occupying a position.

Very high stringency conditions: The term “very high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 70° C.

Very low stringency conditions: The term “very low stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 45° C.

DETAILED DESCRIPTION OF THE INVENTION

Polypeptides Having Lipase Activity

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 2 ofat least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%, which have lipaseactivity. In one aspect, the polypeptides differ by no more than 24amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, or 24 from the mature polypeptide of SEQID NO: 2.

A polypeptide of the present invention preferably comprises or consistsof the amino acid sequence of SEQ ID NO: 2 or an allelic variantthereof; or is a fragment thereof having lipase activity. In anotheraspect, the polypeptide comprises or consists of the mature polypeptideof SEQ ID NO: 2. In another aspect, the polypeptide comprises orconsists of amino acids 1 to 292 of SEQ ID NO: 2.

In another embodiment, the present invention relates to an isolatedpolypeptide having lipase activity encoded by a polynucleotide thathybridizes under very low stringency conditions, low stringencyconditions, medium stringency conditions, medium-high stringencyconditions, high stringency conditions, or very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, or (ii) the full-length complement of (i) (Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor,N.Y.).

The polynucleotide of SEQ ID NO: 1 or a subsequence thereof, as well asthe polypeptide of SEQ ID NO: 2 or a fragment thereof, may be used todesign nucleic acid probes to identify and clone DNA encodingpolypeptides having lipase activity from strains of different genera orspecies according to methods well known in the art. In particular, suchprobes can be used for hybridization with the genomic DNA or cDNA of acell of interest, following standard Southern blotting procedures, inorder to identify and isolate the corresponding gene therein. Suchprobes can be considerably shorter than the entire sequence, but shouldbe at least 15, e.g., at least 25, at least 35, or at least 70nucleotides in length. Preferably, the nucleic acid probe is at least100 nucleotides in length, e.g., at least 200 nucleotides, at least 300nucleotides, at least 400 nucleotides, at least 500 nucleotides, atleast 600 nucleotides, at least 700 nucleotides, at least 800nucleotides, or at least 900 nucleotides in length. Both DNA and RNAprobes can be used. The probes are typically labeled for detecting thecorresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin).Such probes are encompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a polypeptide having lipase activity. Genomic or other DNA fromsuch other strains may be separated by agarose or polyacrylamide gelelectrophoresis, or other separation techniques. DNA from the librariesor the separated DNA may be transferred to and immobilized onnitrocellulose or other suitable carrier material. In order to identifya clone or DNA that hybridizes with SEQ ID NO: 1 or a subsequencethereof, the carrier material is used in a Southern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto (i) SEQ ID NO: 1; (ii) the mature polypeptide coding sequence of SEQID NO: 1; (iii) the full-length complement thereof; or (v) a subsequencethereof; under very low to very high stringency conditions. Molecules towhich the nucleic acid probe hybridizes under these conditions can bedetected using, for example, X-ray film or any other detection meansknown in the art.

In another embodiment, the present invention relates to an isolatedpolypeptide having lipase activity encoded by a polynucleotide having asequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 at least 96%, at least 97%, at least 98%, at least 99%, or 100%.

In another embodiment, the present invention relates to variants of themature polypeptide of SEQ ID NO: 2 comprising a substitution, deletion,and/or insertion at one or more (e.g., several) positions. In anotherembodiment, the present invention relates to variants of the polypeptideof the invention comprising a substitution, deletion, and/or insertionat one or more (e.g., several) positions. In an embodiment, the numberof amino acid substitutions, deletions and/or insertions introduced intothe mature polypeptide of SEQ ID NO: 2 is not more than 24, e.g., 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, or 24. The amino acid changes may be of a minor nature, that isconservative amino acid substitutions or insertions that do notsignificantly affect the folding and/or activity of the protein; smalldeletions, typically of 1-30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to 20-25 residues; or a smallextension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for lipase activity to identify amino acid residuesthat are critical to the activity of the molecule. See also, Hilton etal., 1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzymeor other biological interaction can also be determined by physicalanalysis of structure, as determined by such techniques as nuclearmagnetic resonance, crystallography, electron diffraction, orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver etal., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acidscan also be inferred from an alignment with a related polypeptide.Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO95/17413; or WO95/22625. Other methods that can be usedinclude error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The polypeptide may be a fusion polypeptide or cleavable fusionpolypeptide in which another polypeptide is fused at the N-terminus orthe C-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

Sources of Polypeptides Having Lipase Activity

A polypeptide having lipase activity of the present invention may beobtained from microorganisms of any genus. For purposes of the presentinvention, the term “obtained from” as used herein in connection with agiven source shall mean that the polypeptide encoded by a polynucleotideis produced by the source or by a strain in which the polynucleotidefrom the source has been inserted. In one aspect, the polypeptideobtained from a given source is secreted extracellularly.

The polypeptide may be a bacterial polypeptide. For example, thepolypeptide may be a Gram-positive bacterial polypeptide such as aBacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, orStreptomyces polypeptide having lipase activity, or a Gram-negativebacterial polypeptide such as a Campylobacter, Cedecea, E. coli,Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria,Pseudomonas, Salmonella, Serratia, or Ureaplasma polypeptide.

In one aspect, the polypeptide is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide.

In another aspect, the polypeptide is a Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equisubsp. Zooepidemicus polypeptide.

In another aspect, the polypeptide is a Streptomyces achromogenes,Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus,or Streptomyces lividans polypeptide.

In another aspect, the polypeptide is a Cedecea davisae, Cedecealapegei, Cedecea sp 001 (also known as Cedecea species 3), Cedeceaneteri (formerly known as Cedecea species 4 or Cedecea species 002),Cedecea sp 012 (also known as Cedecea species 5), or Cedecea sp-16640polypeptide.

In another aspect, the polypeptide is a Serratia entomophila, Serratiaficaria, Serratia fonticola, Serratia grimesii, Serratia liquefaciens,Serratia marcescens, Serratia odorifera, Serratia plymuthica, Serratiaproteamaculans, Serratia quinivorans, Serratia rubidaea, Serratiasymbiotica polypeptide.

The polypeptide may be a fungal polypeptide. For example, thepolypeptide may be a yeast polypeptide such as a Candida, Kluyveromyces,Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia polypeptide; ora filamentous fungal polypeptide such as an Acremonium, Agaricus,Alternaria, Aspergillus, Aureobasidium, Botryosphaeria, Ceriporiopsis,Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis,Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia,Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex,Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor,Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces,Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea,Verticillium, Volvariella, or Xylaria polypeptide.

In another aspect, the polypeptide is a Saccharomyces carlsbergensis,Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomycesdouglasii, Saccharomyces kluyveri, Saccharomyces norbensis, orSaccharomyces oviformis polypeptide.

In another aspect, the polypeptide is an Acremonium cellulolyticus,Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus,Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Chrysosporium inops,Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporiummerdarium, Chrysosporium pannicola, Chrysosporium queenslandicum,Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurosporacrassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaetechrysosporium, Thielavia achromatica, Thielavia albomyces, Thielaviaalbopilosa, Thielavia australeinsis, Thielavia fimeti, Thielaviamicrospora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa,Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris,Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride polypeptide.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The polypeptide may be identified and obtained from other sourcesincluding microorganisms isolated from nature (e.g., soil, composts,water, etc.) or DNA samples obtained directly from natural materials(e.g., soil, composts, water, etc.) using the above-mentioned probes.Techniques for isolating microorganisms and DNA directly from naturalhabitats are well known in the art. A polynucleotide encoding thepolypeptide may then be obtained by similarly screening a genomic DNA orcDNA library of another microorganism or mixed DNA sample. Once apolynucleotide encoding a polypeptide has been detected with theprobe(s), the polynucleotide can be isolated or cloned by utilizingtechniques that are known to those of ordinary skill in the art (see,e.g., Sambrook et al., 1989, supra).

Polynucleotides

The present invention also relates to isolated polynucleotides encodinga polypeptide of the present invention, as described herein.

The techniques used to isolate or clone a polynucleotide are known inthe art and include isolation from genomic DNA or cDNA, or a combinationthereof. The cloning of the polynucleotides from genomic DNA can beeffected, e.g., by using the well known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligation activated transcription (LAT) andpolynucleotide-based amplification (NASBA) may be used. Thepolynucleotides may be cloned from a strain of Cedecea, or a relatedorganism and thus, for example, may be an allelic or species variant ofthe polypeptide encoding region of the polynucleotide.

Modification of a polynucleotide encoding a polypeptide of the presentinvention may be necessary for synthesizing polypeptides substantiallysimilar to the polypeptide. The term “substantially similar” to thepolypeptide refers to non-naturally occurring forms of the polypeptide.These polypeptides may differ in some engineered way from thepolypeptide isolated from its native source, e.g., variants that differin specific activity, thermostability, pH optimum, or the like. Thevariants may be constructed on the basis of the polynucleotide presentedas the mature polypeptide coding sequence of SEQ ID NO: 1, e.g., asubsequence thereof, and/or by introduction of nucleotide substitutionsthat do not result in a change in the amino acid sequence of thepolypeptide, but which correspond to the codon usage of the hostorganism intended for production of the enzyme, or by introduction ofnucleotide substitutions that may give rise to a different amino acidsequence. For a general description of nucleotide substitution, see,e.g., Ford et al., 1991, Protein Expression and Purification 2: 95-107.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the expression of the coding sequence in asuitable host cell under conditions compatible with the controlsequences.

A polynucleotide may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the polynucleotide priorto its insertion into a vector may be desirable or necessary dependingon the expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide that isrecognized by a host cell for expression of a polynucleotide encoding apolypeptide of the present invention. The promoter containstranscriptional control sequences that mediate the expression of thepolypeptide. The promoter may be any polynucleotide that showstranscriptional activity in the host cell including mutant, truncated,and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xyIA and xyIB genes,Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO96/00787), Fusariumvenenatum amyloglucosidase (WO00/56900), Fusarium venenatum Daria(WO00/56900), Fusarium venenatum Quinn (WO00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a modified promoter from an Aspergillus neutral alpha-amylasegene in which the untranslated leader has been replaced by anuntranslated leader from an Aspergillus triose phosphate isomerase gene;non-limiting examples include modified promoters from an Aspergillusniger neutral alpha-amylase gene in which the untranslated leader hasbeen replaced by an untranslated leader from an Aspergillus nidulans orAspergillus oryzae triose phosphate isomerase gene); and mutant,truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminator isoperably linked to the 3′-terminus of the polynucleotide encoding thepolypeptide. Any terminator that is functional in the host cell may beused in the present invention.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans anthranilate synthase,Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase,Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-likeprotease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO94/25612) and a Bacillus subtilisSP82 gene (Hue et al., 1995, Journal of Bacteriology 177: 3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leader isoperably linked to the 5′-terminus of the polynucleotide encoding thepolypeptide. Any leader that is functional in the host cell may be used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. A foreign signal peptide coding sequence may be required wherethe coding sequence does not naturally contain a signal peptide codingsequence. Alternatively, a foreign signal peptide coding sequence maysimply replace the natural signal peptide coding sequence in order toenhance secretion of the polypeptide. However, any signal peptide codingsequence that directs the expressed polypeptide into the secretorypathway of a host cell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of apolypeptide and the signal peptide sequence is positioned next to theN-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the polypeptide relative to the growth of the host cell.Examples of regulatory systems are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysystems in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used. Other examples of regulatorysequences are those that allow for gene amplification. In eukaryoticsystems, these regulatory sequences include the dihydrofolate reductasegene that is amplified in the presence of methotrexate, and themetallothionein genes that are amplified with heavy metals. In thesecases, the polynucleotide encoding the polypeptide would be operablylinked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleotideand control sequences may be joined together to produce a recombinantexpression vector that may include one or more convenient restrictionsites to allow for insertion or substitution of the polynucleotideencoding the polypeptide at such sites. Alternatively, thepolynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin, or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a polypeptide. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the production of a polypeptide of thepresent invention. A construct or vector comprising a polynucleotide isintroduced into a host cell so that the construct or vector ismaintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thepolypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacifius, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, Cedecea, E. coli, Flavobacterium, Fusobacterium,Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, Serratia,and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell including, butnot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The bacterial host cell may also be any Cedecea cell including, but notlimited to, Cedecea devisee, Cedecea lapegei, Cedecea sp 001 (also knownas Cedecea species 3), Cedecea neteri (formerly known as Cedecea species4 or Cedecea species 002), Cedecea sp 012 (also known as Cedecea species5), or Cedecea sp-16640 polypeptide.

The bacterial host cell may also be any Serratia cell including, but notlimited to, Serratia entomophila, Serratia ficaria, Serratia fonticola,Serratia grimesii, Serratia liquefaciens, Serratia marcescens, Serratiaodorifera, Serratia plymuthica, Serratia proteamaculans, Serratiaquinivorans, Serratia rubidaea, Serratia symbiotica polypeptide.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (e.g., Mazodier et al., 1989, J. Bacteriol. 171: 3583-3585),or transduction (e.g., Burke et al., 2001, Proc. Natl. Acad. Sci. USA98: 6289-6294). The introduction of DNA into a Pseudomonas cell may beeffected by electroporation (see, e.g., Choi et al., 2006, J. Microbiol.Methods 64: 391-397) or conjugation (e.g., Pinedo and Smets, 2005, Appl.Environ. Microbiol. 71: 51-57). The introduction of DNA into aStreptococcus cell may be effected by natural competence (see, e.g.,Perry and Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), protoplasttransformation (e.g., Catt and Jollick, 1991, Microbios 68: 189-207),electroporation (e.g., Buckley et al., 1999, Appl. Environ. Microbiol.65: 3800-3804), or conjugation (e.g., Clewell, 1981, Microbiol. Rev. 45:409-436). However, any method known in the art for introducing DNA intoa host cell can be used.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a polypeptideof the present invention, comprising (a) cultivating a cell, which inits wild-type form produces the polypeptide, under conditions conducivefor production of the polypeptide; and (b) recovering the polypeptide.In a preferred aspect, the cell is a Cedecea cell. In a more preferredaspect, the cell is a Cedecea sp-16640 cell.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising (a) cultivating a recombinant hostcell of the present invention under conditions conducive for productionof the polypeptide; and (b) recovering the polypeptide.

The host cells are cultivated in a nutrient medium suitable forproduction of the polypeptide using methods known in the art. Forexample, the cell may be cultivated by shake flask cultivation, orsmall-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe polypeptide to be expressed and/or isolated. The cultivation takesplace in a suitable nutrient medium comprising carbon and nitrogensources and inorganic salts, using procedures known in the art. Suitablemedia are available from commercial suppliers or may be preparedaccording to published compositions (e.g., in catalogues of the AmericanType Culture Collection). If the polypeptide is secreted into thenutrient medium, the polypeptide can be recovered directly from themedium. If the polypeptide is not secreted, it can be recovered fromcell lysates.

The polypeptide may be detected using methods known in the art that arespecific for the polypeptides. These detection methods include, but arenot limited to, use of specific antibodies, formation of an enzymeproduct, or disappearance of an enzyme substrate. For example, an enzymeassay may be used to determine the activity of the polypeptide.

The polypeptide may be recovered using methods known in the art. Forexample, the polypeptide may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptide may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure polypeptides.

In an alternative aspect, the polypeptide is not recovered, but rather ahost cell of the present invention expressing the polypeptide is used asa source of the polypeptide.

Plants

The present invention also relates to isolated plants, e.g., atransgenic plant, plant part, or plant cell, comprising a polynucleotideof the present invention so as to express and produce a polypeptide ordomain in recoverable quantities. The polypeptide or domain may berecovered from the plant or plant part. Alternatively, the plant orplant part containing the polypeptide or domain may be used as such forimproving the quality of a food or feed, e.g., improving nutritionalvalue, palatability, and rheological properties, or to destroy anantinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilization of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seed coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing the polypeptide or domainmay be constructed in accordance with methods known in the art. Inshort, the plant or plant cell is constructed by incorporating one ormore expression constructs encoding the polypeptide or domain into theplant host genome or chloroplast genome and propagating the resultingmodified plant or plant cell into a transgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct thatcomprises a polynucleotide encoding a polypeptide or domain operablylinked with appropriate regulatory sequences required for expression ofthe polynucleotide in the plant or plant part of choice. Furthermore,the expression construct may comprise a selectable marker useful foridentifying plant cells into which the expression construct has beenintegrated and DNA sequences necessary for introduction of the constructinto the plant in question (the latter depends on the DNA introductionmethod to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences, is determined, forexample, on the basis of when, where, and how the polypeptide or domainis desired to be expressed. For instance, the expression of the geneencoding a polypeptide or domain may be constitutive or inducible, ormay be developmental, stage or tissue specific, and the gene product maybe targeted to a specific tissue or plant part such as seeds or leaves.Regulatory sequences are, for example, described by Tague et al., 1988,Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, or therice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689; Zhanget al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant Cell Physiol. 39: 885-889), a Vicia faba promoter fromthe legumin B4 and the unknown seed protein gene from Vicia faba (Conradet al., 1998, J. Plant Physiol. 152: 708-711), a promoter from a seedoil body protein (Chen et al., 1998, Plant Cell Physiol. 39: 935-941),the storage protein napA promoter from Brassica napus, or any other seedspecific promoter known in the art, e.g., as described in WO91/14772.Furthermore, the promoter may be a leaf specific promoter such as therbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiol.102: 991-1000), the chlorella virus adenine methyltransferase genepromoter (Mitra and Higgins, 1994, Plant Mol. Biol. 26: 85-93), the aldPgene promoter from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248:668-674), or a wound inducible promoter such as the potato pin2 promoter(Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promotermay be induced by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a polypeptide or domain in the plant. For instance, thepromoter enhancer element may be an intron that is placed between thepromoter and the polynucleotide encoding a polypeptide or domain. Forinstance, Xu et al., 1993, supra, disclose the use of the first intronof the rice actin 1 gene to enhance expression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Agrobacterium tumefaciens-mediated gene transfer is a method forgenerating transgenic dicots (for a review, see Hooykas andSchilperoort, 1992, Plant Mol. Biol. 19: 15-38) and for transformingmonocots, although other transformation methods may be used for theseplants. A method for generating transgenic monocots is particlebombardment (microscopic gold or tungsten particles coated with thetransforming DNA) of embryonic calli or developing embryos (Christou,1992, Plant J. 2: 275-281; Shimamoto, 1994, Curr. Opin. Biotechnol. 5:158-162; Vasil et al., 1992, Bio/Technology 10: 667-674). An alternativemethod for transformation of monocots is based on protoplasttransformation as described by Omirulleh et al., 1993, Plant Mol. Biol.21: 415-428. Additional transformation methods include those describedin U.S. Pat. No. 6,395,966 and U.S. Pat. No. 7,151,204 (both of whichare herein incorporated by reference in their entirety).

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

In addition to direct transformation of a particular plant genotype witha construct of the present invention, transgenic plants may be made bycrossing a plant having the construct to a second plant lacking theconstruct. For example, a construct encoding a polypeptide or domain canbe introduced into a particular plant variety by crossing, without theneed for ever directly transforming a plant of that given variety.Therefore, the present invention encompasses not only a plant directlyregenerated from cells which have been transformed in accordance withthe present invention, but also the progeny of such plants. As usedherein, progeny may refer to the offspring of any generation of a parentplant prepared in accordance with the present invention. Such progenymay include a DNA construct prepared in accordance with the presentinvention. Crossing results in the introduction of a transgene into aplant line by cross pollinating a starting line with a donor plant line.Non-limiting examples of such steps are described in U.S. Pat. No.7,151,204.

Plants may be generated through a process of backcross conversion. Forexample, plants include plants referred to as a backcross convertedgenotype, line, inbred, or hybrid.

Genetic markers may be used to assist in the introgression of one ormore transgenes of the invention from one genetic background intoanother. Marker assisted selection offers advantages relative toconventional breeding in that it can be used to avoid errors caused byphenotypic variations. Further, genetic markers may provide dataregarding the relative degree of elite germplasm in the individualprogeny of a particular cross. For example, when a plant with a desiredtrait which otherwise has a non-agronomically desirable geneticbackground is crossed to an elite parent, genetic markers may be used toselect progeny which not only possess the trait of interest, but alsohave a relatively large proportion of the desired germplasm. In thisway, the number of generations required to introgress one or more traitsinto a particular genetic background is minimized.

The present invention also relates to methods of producing a polypeptideor domain of the present invention comprising (a) cultivating atransgenic plant or a plant cell comprising a polynucleotide encodingthe polypeptide or domain under conditions conducive for production ofthe polypeptide or domain; and (b) recovering the polypeptide or domain.

Compositions

The non-limiting list of composition components illustrated hereinafterare suitable for use in the compositions and methods herein may bedesirably incorporated in certain embodiments of the invention, e.g. toassist or enhance cleaning performance, for treatment of the substrateto be cleaned, or to modify the aesthetics of the composition as is thecase with perfumes, colorants, dyes or the like. The levels of any suchcomponents incorporated in any compositions are in addition to anymaterials previously recited for incorporation. The precise nature ofthese additional components, and levels of incorporation thereof, willdepend on the physical form of the composition and the nature of thecleaning operation for which it is to be used. Unless otherwiseindicated the amounts in percentage is by weight of the composition (wt%). Suitable component materials include, but are not limited to,surfactants, builders, chelating agents, dye transfer inhibiting agents,dispersants, enzymes, and enzyme stabilizers, catalytic materials,bleach activators, hydrogen peroxide, sources of hydrogen peroxide,preformed peracids, polymeric dispersing agents, clay soilremoval/anti-redeposition agents, brighteners, suds suppressors, dyes,hueing dyes, perfumes, perfume delivery systems, structure elasticizingagents, fabric softeners, carriers, hydrotropes, processing aids,solvents and/or pigments. In addition to the disclosure below, suitableexamples of such other components and levels of use are found in U.S.Pat. No. 5,576,282; U.S. Pat. No. 6,306,812; and U.S. Pat. No. 6,326,348hereby incorporated by reference.

Thus, in certain embodiments the invention do not contain one or more ofthe following adjuncts materials: surfactants, builders, chelatingagents, dye transfer inhibiting agents, dispersants, additional enzymes,and enzyme stabilizers, catalytic materials, bleach activators, hydrogenperoxide, sources of hydrogen peroxide, preformed peracids, polymericdispersing agents, clay soil removal/anti-redeposition agents,brighteners, suds suppressors, dyes, perfumes, perfume delivery systems,structure elasticizing agents, fabric softeners, carriers, hydrotropes,processing aids, solvents and/or pigments. However, when one or morecomponents are present, such one or more components may be present asdetailed below:

Surfactants—

The compositions according to the present invention may comprise asurfactant or surfactant system wherein the surfactant can be selectedfrom nonionic surfactants, anionic surfactants, cationic surfactants,ampholytic surfactants, zwitterionic surfactants, semi-polar nonionicsurfactants and mixtures thereof. When present, surfactant is typicallypresent at a level of from 0.1 to 60 wt %, from 1 to 50 wt % or from 5to 40 wt %.

Suitable anionic detersive surfactants include sulphate and sulphonatedetersive surfactants.

Suitable sulphonate detersive surfactants include alkyl benzenesulphonate, in one aspect, C₁₀₋₁₃ alkyl benzene sulphonate. Suitablealkyl benzene sulphonate (LAS) may be obtained, by sulphonatingcommercially available linear alkyl benzene (LAB); suitable LAB includeslow 2-phenyl LAB, such as Isochem® or Petrelab®, other suitable LABinclude high 2-phenyl LAB, such as Hyblene®. A suitable anionicdetersive surfactant is alkyl benzene sulphonate that is obtained byDETAL catalyzed process, although other synthesis routes, such as HF,may also be suitable. In one aspect a magnesium salt of LAS is used.

Suitable sulphate detersive surfactants include alkyl sulphate, in oneaspect, C₈₋₁₈ alkyl sulphate, or predominantly C₁₂ alkyl sulphate.

Another suitable sulphate detersive surfactant is alkyl alkoxylatedsulphate, in one aspect, alkyl ethoxylated sulphate, in one aspect, aC₈₋₁₈ alkyl alkoxylated sulphate, in another aspect, a C₈₋₁₈ alkylethoxylated sulphate, typically the alkyl alkoxylated sulphate has anaverage degree of alkoxylation of from 0.5 to 20, or from 0.5 to 10,typically the alkyl alkoxylated sulphate is a C₈₋₁₈ alkyl ethoxylatedsulphate having an average degree of ethoxylation of from 0.5 to 10,from 0.5 to 7, from 0.5 to 5 or from 0.5 to 3.

The alkyl sulphate, alkyl alkoxylated sulphate and alkyl benzenesulphonates may be linear or branched, substituted or un-substituted.

The detersive surfactant may be a mid-chain branched detersivesurfactant, in one aspect, a mid-chain branched anionic detersivesurfactant, in one aspect, a mid-chain branched alkyl sulphate and/or amid-chain branched alkyl benzene sulphonate, e.g. a mid-chain branchedalkyl sulphate. In one aspect, the mid-chain branches are C₁₋₄ alkylgroups, typically methyl and/or ethyl groups.

Suitable non-ionic detersive surfactants are selected from the groupconsisting of: C₈-C₁₈ alkyl ethoxylates, such as, NEODOL®; C₆-C₁₂ alkylphenol alkoxylates wherein the alkoxylate units may be ethyleneoxyunits, propyleneoxy units or a mixture thereof; C₁₂-C₁₈ alcohol andC₆-C₁₂ alkyl phenol condensates with ethylene oxide/propylene oxideblock polymers such as Pluronic®; C₁₄-C₂₂ mid-chain branched alcohols;C₁₄-C₂₂ mid-chain branched alkyl alkoxylates, typically having anaverage degree of alkoxylation of from 1 to 30; alkylpolysaccharides, inone aspect, alkylpolyglycosides; polyhydroxy fatty acid amides; ethercapped poly(oxyalkylated) alcohol surfactants; and mixtures thereof.

Suitable non-ionic detersive surfactants include alkyl polyglucosideand/or an alkyl alkoxylated alcohol.

In one aspect, non-ionic detersive surfactants include alkyl alkoxylatedalcohols, in one aspect C₈₋₁₈ alkyl alkoxylated alcohol, e.g. a C₈₋₁₈alkyl ethoxylated alcohol, the alkyl alkoxylated alcohol may have anaverage degree of alkoxylation of from 1 to 50, from 1 to 30, from 1 to20, or from 1 to 10.

In one aspect, the alkyl alkoxylated alcohol may be a C₈₋₁₈ alkylethoxylated alcohol having an average degree of ethoxylation of from 1to 10, from 1 to 7, more from 1 to 5 or from 3 to 7. The alkylalkoxylated alcohol can be linear or branched, and substituted orun-substituted. Suitable nonionic surfactants include Lutensol®.

Suitable cationic detersive surfactants include alkyl pyridiniumcompounds, alkyl quaternary ammonium compounds, alkyl quaternaryphosphonium compounds, alkyl ternary sulphonium compounds, and mixturesthereof.

Suitable cationic detersive surfactants are quaternary ammoniumcompounds having the general formula: (R)(R₁)(R₂)(R₃)N⁺X⁻, wherein, R isa linear or branched, substituted or unsubstituted C₆₋₁₈ alkyl oralkenyl moiety, R₁ and R₂ are independently selected from methyl orethyl moieties, R₃ is a hydroxyl, hydroxymethyl or a hydroxyethylmoiety, X is an anion which provides charge neutrality, suitable anionsinclude: halides, e.g. chloride; sulphate; and sulphonate. Suitablecationic detersive surfactants are mono-C₆₋₁₈ alkyl mono-hydroxyethyldi-methyl quaternary ammonium chlorides. Highly suitable cationicdetersive surfactants are mono-C₈₋₁₀ alkyl mono-hydroxyethyl di-methylquaternary ammonium chloride, mono-C₁₀₋₁₂ alkyl mono-hydroxyethyldi-methyl quaternary ammonium chloride and mono-C₁₀ alkylmono-hydroxyethyl di-methyl quaternary ammonium chloride.

Suitable amphoteric/zwitterionic surfactants include amine oxides andbetaines. Amine-neutralized anionic surfactants—Anionic surfactants ofthe present invention and adjunct anionic cosurfactants, may exist in anacid form, and said acid form may be neutralized to form a surfactantsalt which is desirable for use in the present detergent compositions.Typical agents for neutralization include the metal counterion base suchas hydroxides, eg, NaOH or KOH. Further preferred agents forneutralizing anionic surfactants of the present invention and adjunctanionic surfactants or cosurfactants in their acid forms includeammonia, amines, or alkanolamines. Alkanolamines are preferred. Suitablenon-limiting examples including monoethanolamine, diethanolamine,triethanolamine, and other linear or branched alkanolamines known in theart; e.g., highly preferred alkanolamines include 2-amino-1-propanol,1-aminopropanol, monoisopropanolamine, or 1-amino-3-propanol. Amineneutralization may be done to a full or partial extent, e.g. part of theanionic surfactant mix may be neutralized with sodium or potassium andpart of the anionic surfactant mix may be neutralized with amines oralkanolamines.

Surfactant systems comprising mixtures of one or more anionic and inaddition one or more nonionic surfactants optionally with an additionalsurfactant such as a cationic surfactant, may be preferred. Preferredweight ratios of anionic to nonionic surfactant are at least 2:1, or atleast 1:1 to 1:10.

Builders—

The compositions of the present invention may comprise one or morebuilders, co-builders, builder systems or a mixture thereof. When abuilder is used, the cleaning composition will typically comprise atleast 1 wt %, from 2 to 60 wt % or from 5 to 10 wt % builder. In a dishwash cleaning composition, the level of builder is typically 40 to 65 wt%, or 50 to 65 wt %. The composition may be substantially free ofbuilder; substantially free means “no deliberately added” zeolite and/orphosphate. Typical zeolite builders include zeolite A, zeolite P andzeolite MAP. A typical phosphate builder is sodium tri-polyphosphate.

The builder and/or co-builder may particularly be a chelating agent thatforms water-soluble complexes with Ca and Mg. Any builder and/orco-builder known in the art for use in detergents may be utilized.Non-limiting examples of builders include zeolites, diphosphates(pyrophosphates), triphosphates such as sodium triphosphate (STP orSTPP), carbonates such as sodium carbonate, soluble silicates such assodium metasilicate, layered silicates (e.g., SKS-6 from Hoechst),ethanolamines such as 2-aminoethan-1-ol (MEA), iminodiethanol (DEA) and2,2′,2″-nitrilotriethanol (TEA), and carboxymethylinulin (CMI), andcombinations thereof.

The cleaning composition may include a co-builder alone, or incombination with a builder, e.g. a zeolite builder. Non-limitingexamples of co-builders include homopolymers of polyacrylates orcopolymers thereof, such as poly(acrylic acid) (PAA) or copoly(acrylicacid/maleic acid) (PAA/PMA). Further non-limiting examples includecitrate, chelators such as aminocarboxylates, aminopolycarboxylates andphosphonates, and alkyl- or alkenylsuccinic acid. Additional specificexamples include 2,2′,2″-nitrilotriacetic acid (NTA),etheylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N′-disuccinicacid (EDDS), methylglycinediacetic acid (MGDA), glutamicacid-N,N-diacetic acid (GLDA), 1-hydroxyethane-1,1-diylbis(phosphonicacid) (HEDP), ethylenediaminetetrakis(methylene)tetrakis(phosphonicacid) (EDTMPA), diethylenetriaminepentakis(methylene)pentakis(phosphonicacid) (DTPMPA), N-(2-hydroxyethyl)iminodiacetic acid (EDG), asparticacid-N-monoacetic acid (ASMA), aspartic acid-N,N-diacetic acid (ASDA),aspartic acid-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA),N-(2-sulfomethyl) aspartic acid (SMAS), N-(2-sulfoethyl) aspartic acid(SEAS), N-(2-sulfomethyl) glutamic acid (SMGL), N-(2-sulfoethyl)glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA),α-alanine-N,N-diacetic acid (α-ALDA), serine-N,N-diacetic acid (SEDA),isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid(PHDA), anthranilic acid-N,N-diacetic acid (ANDA), sulfanilic acid-N,N-diacetic acid (SLDA), taurine-N, N-diacetic acid (TUDA) andsulfomethyl-N,N-diacetic acid (SMDA),N-(hydroxyethyl)-ethylidenediaminetriacetate (HEDTA), diethanolglycine(DEG), Diethylenetriamine Penta (Methylene Phosphonic acid) (DTPMP),aminotris(methylenephosphonic acid) (ATMP), and combinations and saltsthereof. Further exemplary builders and/or co-builders are described in,e.g., WO09/102854, U.S. Pat. No. 5,977,053.

Chelating Agents and Crystal Growth Inhibitors—

The compositions herein may contain a chelating agent and/or a crystalgrowth inhibitor. Suitable molecules include copper, iron and/ormanganese chelating agents and mixtures thereof. Suitable moleculesinclude DTPA (Diethylene triamine pentaacetic acid), HEDP (Hydroxyethanediphosphonic acid), DTPMP (Diethylene triamine penta(methylenephosphonic acid)), 1,2-Dihydroxybenzene-3,5-disulfonic acid disodiumsalt hydrate, ethylenediamine, diethylene triamine,ethylenediaminedisuccinic acid (EDDS),N-hydroxyethylethylenediaminetri-acetic acid (HEDTA),triethylenetetraaminehexaacetic acid (TTHA), N-hydroxyethyliminodiaceticacid (HEIDA), dihydroxyethylglycine (DHEG),ethylenediaminetetrapropionic acid (EDTP), carboxymethyl inulin and2-Phosphonobutane 1,2,4-tricarboxylic acid (Bayhibit® AM) andderivatives thereof. Typically the composition may comprise from 0.005to 15 wt % or from 3.0 to 10 wt % chelating agent or crystal growthinhibitor.

Bleach Component—

The bleach component suitable for incorporation in the methods andcompositions of the invention comprise one or a mixture of more than onebleach component. Suitable bleach components include bleachingcatalysts, photobleaches, bleach activators, hydrogen peroxide, sourcesof hydrogen peroxide, pre-formed peracids and mixtures thereof. Ingeneral, when a bleach component is used, the compositions of thepresent invention may comprise from 0.00001 to 90 wt %, 0.0001 to 50 wt% or from 0.001 to 25 wt %. Examples of suitable bleach componentsinclude:

(1) Pre-formed peracids: Suitable preformed peracids include, but arenot limited to, compounds selected from the group consisting ofpre-formed peroxyacids or salts thereof, typically either aperoxycarboxylic acid or salt thereof, or a peroxysulphonic acid or saltthereof.

The pre-formed peroxyacid or salt thereof is preferably aperoxycarboxylic acid or salt thereof, typically having a chemicalstructure corresponding to the following chemical formula:

wherein: R¹⁴ is selected from alkyl, aralkyl, cycloalkyl, aryl orheterocyclic groups; the R¹⁴ group can be linear or branched,substituted or unsubstituted; and Y is any suitable counter-ion thatachieves electric charge neutrality, preferably Y is selected fromhydrogen, sodium or potassium. Preferably, R¹⁴ is a linear or branched,substituted or unsubstituted C₆₋₉ alkyl. Preferably, the peroxyacid orsalt thereof is selected from peroxyhexanoic acid, peroxyheptanoic acid,peroxyoctanoic acid, peroxynonanoic acid, peroxydecanoic acid, any saltthereof, or any combination thereof. Particularly preferred peroxyacidsare phthalimido-peroxy-alkanoic acids, in particular ε-phthahlimidoperoxy hexanoic acid (PAP). Preferably, the peroxyacid or salt thereofhas a melting point in the range of from 30° C. to 60° C.

The pre-formed peroxyacid or salt thereof can also be a peroxysulphonicacid or salt thereof, typically having a chemical structurecorresponding to the following chemical formula:

wherein: R¹⁵ is selected from alkyl, aralkyl, cycloalkyl, aryl orheterocyclic groups; the R¹⁵ group can be linear or branched,substituted or unsubstituted; and Z is any suitable counter-ion thatachieves electric charge neutrality, preferably Z is selected fromhydrogen, sodium or potassium. Preferably R¹⁵ is a linear or branched,substituted or unsubstituted C₆₋₉ alkyl. Preferably such bleachcomponents may be present in the compositions of the invention in anamount from 0.01 to 50 wt % or from 0.1 to 20 wt %.

(2) Sources of hydrogen peroxide include e.g., inorganic perhydratesalts, including alkali metal salts such as sodium salts of perborate(usually mono- or tetra-hydrate), percarbonate, persulphate,perphosphate, persilicate salts and mixtures thereof. In one aspect ofthe invention the inorganic perhydrate salts such as those selected fromthe group consisting of sodium salts of perborate, percarbonate andmixtures thereof. When employed, inorganic perhydrate salts aretypically present in amounts of 0.05 to 40 wt % or 1 to 30 wt % of theoverall composition and are typically incorporated into suchcompositions as a crystalline solid that may be coated. Suitablecoatings include: inorganic salts such as alkali metal silicate,carbonate or borate salts or mixtures thereof, or organic materials suchas water-soluble or dispersible polymers, waxes, oils or fatty soaps.Preferably such bleach components may be present in the compositions ofthe invention in an amount of 0.01 to 50 wt % or 0.1 to 20 wt %.

(3) Suitable bleach activators are those having R—(C═O)-L wherein R isan alkyl group, optionally branched, having, when the bleach activatoris hydrophobic, from 6 to 14 carbon atoms, or from 8 to 12 carbon atomsand, when the bleach activator is hydrophilic, less than 6 carbon atomsor less than 4 carbon atoms; and L is leaving group. Examples ofsuitable leaving groups are benzoic acid and derivativesthereof—especially benzene sulphonate. Suitable bleach activatorsinclude dodecanoyl oxybenzene sulphonate, decanoyl oxybenzenesulphonate, decanoyl oxybenzoic acid or salts thereof, 3,5,5-trimethylhexanoyloxybenzene sulphonate, tetraacetyl ethylene diamine (TAED) andnonanoyloxybenzene sulphonate (NOBS). Suitable bleach activators arealso disclosed in WO98/17767. While any suitable bleach activator may beemployed, in one aspect of the invention the subject cleaningcomposition may comprise NOBS, TAED or mixtures thereof. When present,the peracid and/or bleach activator is generally present in thecomposition in an amount of 0.1 to 60 wt %, 0.5 to 40 wt % or 0.6 to 10wt % based on the fabric and home care composition. One or morehydrophobic peracids or precursors thereof may be used in combinationwith one or more hydrophilic peracid or precursor thereof. Preferablysuch bleach components may be present in the compositions of theinvention in an amount of 0.01 to 50 wt %, or 0.1 to 20 wt %.

The amounts of hydrogen peroxide source and peracid or bleach activatormay be selected such that the molar ratio of available oxygen (from theperoxide source) to peracid is from 1:1 to 35:1, or even 2:1 to 10:1.

(4) Diacyl peroxides—preferred diacyl peroxide bleaching species includethose selected from diacyl peroxides of the general formula:R¹—C(O)—OO—(O)C—R², in which R¹ represents a C₆-C₁₈ alkyl, preferablyC₆-C₁₂ alkyl group containing a linear chain of at least 5 carbon atomsand optionally containing one or more substituents (e.g. —N⁺ (CH₃)₃,—COOH or —CN) and/or one or more interrupting moieties (e.g. —CONH— or—CH═CH—) interpolated between adjacent carbon atoms of the alkylradical, and R² represents an aliphatic group compatible with a peroxidemoiety, such that R¹ and R² together contain a total of 8 to 30 carbonatoms. In one preferred aspect R¹ and R² are linear unsubstituted C₆-C₁₂alkyl chains. Most preferably R¹ and R² are identical. Diacyl peroxides,in which both R¹ and R² are C₆-C₁₂ alkyl groups, are particularlypreferred. Preferably, at least one of, most preferably only one of, theR groups (R₁ or R₂), does not contain branching or pendant rings in thealpha position, or preferably neither in the alpha nor beta positions ormost preferably in none of the alpha or beta or gamma positions. In onefurther preferred embodiment the DAP may be asymmetric, such thatpreferably the hydrolysis of R1 acyl group is rapid to generate peracid,but the hydrolysis of R2 acyl group is slow.

The tetraacyl peroxide bleaching species is preferably selected fromtetraacyl peroxides of the general formula:R³—C(O)—OO—C(O)—(CH₂)n-C(O)—OO—C(O)—R³, in which R³ represents a C₁-C₉alkyl, or C₃-C₇, group and n represents an integer from 2 to 12, or 4 to10 inclusive.

Preferably, the diacyl and/or tetraacyl peroxide bleaching species ispresent in an amount sufficient to provide at least 0.5 ppm, at least 10ppm, or at least 50 ppm by weight of the wash liquor. In a preferredembodiment, the bleaching species is present in an amount sufficient toprovide from 0.5 to 300 ppm, from 30 to 150 ppm by weight of the washliquor.

Preferably the bleach component comprises a bleach catalyst (5 and 6).

(5) Preferred are organic (non-metal) bleach catalysts include bleachcatalyst capable of accepting an oxygen atom from a peroxyacid and/orsalt thereof, and transferring the oxygen atom to an oxidizeablesubstrate. Suitable bleach catalysts include, but are not limited to:iminium cations and polyions; iminium zwitterions; modified amines;modified amine oxides; N-sulphonyl imines; N-phosphonyl imines; N-acylimines; thiadiazole dioxides; perfluoroimines; cyclic sugar ketones andmixtures thereof.

Suitable iminium cations and polyions include, but are not limited to,N-methyl-3,4-dihydroisoquinolinium tetrafluoroborate, prepared asdescribed in Tetrahedron (1992), 49(2), 423-38 (see e.g. compound 4, p.433); N-methyl-3,4-dihydroisoquinolinium p-toluene sulphonate, preparedas described in U.S. Pat. No. 5,360,569 (see e.g. Column 11, Example 1);and N-octyl-3,4-dihydroisoquinolinium p-toluene sulphonate, prepared asdescribed in U.S. Pat. No. 5,360,568 (see e.g. Column 10, Example 3).

Suitable iminium zwitterions include, but are not limited to,N-(3-sulfopropyl)-3,4-dihydroisoquinolinium, inner salt, prepared asdescribed in U.S. Pat. No. 5,576,282 (see e.g. Column 31, Example II);N-[2-(sulphooxy)dodecyl]-3,4-dihydroisoquinolinium, inner salt, preparedas described in U.S. Pat. No. 5,817,614 (see e.g. Column 32, Example V);2-[3-[(2-ethylhexyl)oxy]-2-(sulphooxy)propyl]-3,4-dihydroisoquinolinium,inner salt, prepared as described in WO05/047264 (see e.g. page 18,Example 8), and2-[3-[(2-butyloctyl)oxy]-2-(sulphooxy)propyl]-3,4-dihydroisoquinolinium,inner salt.

Suitable modified amine oxygen transfer catalysts include, but are notlimited to, 1,2,3,4-tetrahydro-2-methyl-1-isoquinolinol, which can bemade according to the procedures described in Tetrahedron Letters(1987), 28(48), 6061-6064. Suitable modified amine oxide oxygen transfercatalysts include, but are not limited to, sodium1-hydroxy-N-oxy-N-[2-(sulphooxy)decyl]-1,2,3,4-tetrahydroisoquinoline.

Suitable N-sulphonyl imine oxygen transfer catalysts include, but arenot limited to, 3-methyl-1,2-benzisothiazole 1,1-dioxide, preparedaccording to the procedure described in the Journal of Organic Chemistry(1990), 55(4), 1254-61.

Suitable N-phosphonyl imine oxygen transfer catalysts include, but arenot limited to,[R-(E)]-N-[(2-chloro-5-nitrophenyl)methylene]-P-phenyl-P-(2,4,6-trimethylphenyl)-phosphinicamide, which can be made according to the procedures described in theJournal of the Chemical Society, Chemical Communications (1994), (22),2569-70.

Suitable N-acyl imine oxygen transfer catalysts include, but are notlimited to, [N(E)]-N-(phenylmethylene)acetamide, which can be madeaccording to the procedures described in Polish Journal of Chemistry(2003), 77(5), 577-590.

Suitable thiadiazole dioxide oxygen transfer catalysts include but arenot limited to, 3-methyl-4-phenyl-1,2,5-thiadiazole 1,1-dioxide, whichcan be made according to the procedures described in U.S. Pat. No.5,753,599 (Column 9, Example 2).

Suitable perfluoroimine oxygen transfer catalysts include, but are notlimited to,(Z)-2,2,3,3,4,4,4-heptafluoro-N-(nonafluorobutyl)butanimidoyl fluoride,which can be made according to the procedures described in TetrahedronLetters (1994), 35(34), 6329-30.

Suitable cyclic sugar ketone oxygen transfer catalysts include, but arenot limited to,1,2:4,5-di-O-isopropylidene-D-erythro-2,3-hexodiuro-2,6-pyranose asprepared in U.S. Pat. No. 6,649,085 (Column 12, Example 1).

The bleach catalyst may comprise an iminium and/or carbonyl functionalgroup and is typically capable of forming an oxaziridinium and/ordioxirane functional group upon acceptance of an oxygen atom, especiallyupon acceptance of an oxygen atom from a peroxyacid and/or salt thereof.The bleach catalyst may comprise an oxaziridinium functional groupand/or is capable of forming an oxaziridinium functional group uponacceptance of an oxygen atom, especially upon acceptance of an oxygenatom from a peroxyacid and/or salt thereof. The bleach catalyst maycomprise a cyclic iminium functional group, preferably wherein thecyclic moiety has a ring size of from five to eight atoms (including thenitrogen atom), preferably six atoms. The bleach catalyst may comprisean aryliminium functional group, preferably a bi-cyclic aryliminiumfunctional group, preferably a 3,4-dihydroisoquinolinium functionalgroup. Typically, the imine functional group is a quaternary iminefunctional group and is typically capable of forming a quaternaryoxaziridinium functional group upon acceptance of an oxygen atom,especially upon acceptance of an oxygen atom from a peroxyacid and/orsalt thereof. In another aspect, the detergent composition comprises ableach component having a log P_(o/w) no greater than 0, no greater than−0.5, no greater than −1.0, no greater than −1.5, no greater than −2.0,no greater than −2.5, no greater than −3.0, or no greater than −3.5. Themethod for determining log P_(o/w) is described in more detail below.

Typically, the bleach ingredient is capable of generating a bleachingspecies having a X_(SO) of from 0.01 to 0.30, from 0.05 to 0.25, or from0.10 to 0.20. The method for determining X_(SO) is described in moredetail below. For example, bleaching ingredients having anisoquinolinium structure are capable of generating a bleaching speciesthat has an oxaziridinium structure. In this example, the X_(SO) is thatof the oxaziridinium bleaching species.

The bleach catalyst may have a chemical structure corresponding to thefollowing chemical formula:

wherein: n and m are independently from 0 to 4, preferably n and m areboth 0; each R¹ is independently selected from a substituted orunsubstituted radical selected from the group consisting of hydrogen,alkyl, cycloalkyl, aryl, fused aryl, heterocyclic ring, fusedheterocyclic ring, nitro, halo, cyano, sulphonato, alkoxy, keto,carboxylic, and carboalkoxy radicals; and any two vicinal R¹substituents may combine to form a fused aryl, fused carbocyclic orfused heterocyclic ring; each R² is independently selected from asubstituted or unsubstituted radical independently selected from thegroup consisting of hydrogen, hydroxy, alkyl, cycloalkyl, alkaryl, aryl,aralkyl, alkylenes, heterocyclic ring, alkoxys, arylcarbonyl groups,carboxyalkyl groups and amide groups; any R² may be joined together withany other of R² to form part of a common ring; any geminal R² maycombine to form a carbonyl; and any two R² may combine to form asubstituted or unsubstituted fused unsaturated moiety; R³ is a C₁ to C₂₀substituted or unsubstituted alkyl; R⁴ is hydrogen or the moietyQ_(t)-A, wherein: Q is a branched or unbranched alkylene, t=0 or 1 and Ais an anionic group selected from the group consisting of OSO₃ ⁻, SO₃ ⁻,CO₂ ⁻, OCO₂ ⁻, OPO₃ ²⁻, OPO₃H⁻ and OPO₂ ⁻; R⁵ is hydrogen or the moiety—CR¹¹R¹²—Y-G_(b)-Y_(c)—[(CR⁹R¹⁰)_(y)—O]_(k)—R⁸, wherein: each Y isindependently selected from the group consisting of O, S, N—H, or N—R⁸;and each R⁸ is independently selected from the group consisting ofalkyl, aryl and heteroaryl, said moieties being substituted orunsubstituted, and whether substituted or unsubsituted said moietieshaving less than 21 carbons; each G is independently selected from thegroup consisting of CO, SO₂, SO, PO and PO₂; R⁹ and R¹⁰ areindependently selected from the group consisting of H and C₁-C₄ alkyl;R¹¹ and R¹² are independently selected from the group consisting of Hand alkyl, or when taken together may join to form a carbonyl; b=0 or 1;c can=0 or 1, but c must=0 if b=0; y is an integer from 1 to 6; k is aninteger from 0 to 20; R⁶ is H, or an alkyl, aryl or heteroaryl moiety;said moieties being substituted or unsubstituted; and X, if present, isa suitable charge balancing counterion, preferably X is present when R⁴is hydrogen, suitable X, include but are not limited to: chloride,bromide, sulphate, methosulphate, sulphonate, p-toluenesulphonate,borontetraflouride and phosphate.

In one embodiment of the present invention, the bleach catalyst has astructure corresponding to general formula below:

wherein R¹³ is a branched alkyl group containing from three to 24 carbonatoms (including the branching carbon atoms) or a linear alkyl groupcontaining from one to 24 carbon atoms; preferably R¹³ is a branchedalkyl group containing from eight to 18 carbon atoms or linear alkylgroup containing from eight to eighteen carbon atoms; preferably R¹³ isselected from the group consisting of 2-propylheptyl, 2-butyloctyl,2-pentylnonyl, 2-hexyldecyl, n-dodecyl, n-tetradecyl, n-hexadecyl,n-octadecyl, iso-nonyl, iso-decyl, iso-tridecyl and iso-pentadecyl;preferably R¹³ is selected from the group consisting of 2-butyloctyl,2-pentylnonyl, 2-hexyldecyl, iso-tridecyl and iso-pentadecyl.

The bleach component may comprise a source of peracid in addition tobleach catalyst, particularly organic bleach catalyst. The source ofperacid may be selected from (a) pre-formed peracid; (b) percarbonate,perborate or persulfate salt (hydrogen peroxide source) preferably incombination with a bleach activator; and (c) perhydrolase enzyme and anester for forming peracid in situ in the presence of water in a textileor hard surface treatment step.

When present, the peracid and/or bleach activator is generally presentin the composition in an amount of from 0.1 to 60 wt %, from 0.5 to 40wt % or from 0.6 to 10 wt % based on the composition. One or morehydrophobic peracids or precursors thereof may be used in combinationwith one or more hydrophilic peracid or precursor thereof.

The amounts of hydrogen peroxide source and peracid or bleach activatormay be selected such that the molar ratio of available oxygen (from theperoxide source) to peracid is from 1:1 to 35:1, or 2:1 to 10:1.

(6) Metal-containing Bleach Catalysts—The bleach component may beprovided by a catalytic metal complex. One type of metal-containingbleach catalyst is a catalyst system comprising a transition metalcation of defined bleach catalytic activity, such as copper, iron,titanium, ruthenium, tungsten, molybdenum, or manganese cations, anauxiliary metal cation having little or no bleach catalytic activity,such as zinc or aluminum cations, and a sequestrate having definedstability constants for the catalytic and auxiliary metal cations,particularly ethylenediaminetetraacetic acid,ethylenediaminetetra(methylenephosphonic acid) and water-soluble saltsthereof. Such catalysts are disclosed in U.S. Pat. No. 4,430,243.Preferred catalysts are described in WO09/839406, U.S. Pat. No.6,218,351 and WO00/012667. Particularly preferred are transition metalcatalyst or ligands therefore that are cross-bridged polydentate N-donorligands.

If desired, the compositions herein can be catalyzed by means of amanganese compound. Such compounds and levels of use are well known inthe art and include, e.g., the manganese-based catalysts disclosed inU.S. Pat. No. 5,576,282.

Cobalt bleach catalysts useful herein are known, and are described e.g.in U.S. Pat. No. 5,597,936; U.S. Pat. No. 5,595,967. Such cobaltcatalysts are readily prepared by known procedures, such as taught e.g.in U.S. Pat. No. 5,597,936 and U.S. Pat. No. 5,595,967.

Compositions herein may also suitably include a transition metal complexof ligands such as bispidones (U.S. Pat. No. 7,501,389) and/ormacropolycyclic rigid ligands—abbreviated as “MRLs”. As a practicalmatter, and not by way of limitation, the compositions and processesherein can be adjusted to provide on the order of at least one part perhundred million of the active MRL species in the aqueous washing medium,and will typically provide from 0.005 to 25 ppm, from 0.05 to 10 ppm, orfrom 0.1 to 5 ppm, of the MRL in the wash liquor.

Suitable transition-metals in the instant transition-metal bleachcatalyst include e.g. manganese, iron and chromium. Suitable MRLsinclude 5,12-diethyl-1,5,8,12-tetraazabicyclo[6.6.2]hexadecane. Suitabletransition metal MRLs are readily prepared by known procedures, such astaught e.g. in U.S. Pat. No. 6,225,464 and WO00/32601.

(7) Photobleaches—suitable photobleaches include e.g. sulfonated zincphthalocyanine sulfonated aluminium phthalocyanines, xanthene dyes andmixtures thereof. Preferred bleach components for use in the presentcompositions of the invention comprise a hydrogen peroxide source,bleach activator and/or organic peroxyacid, optionally generated in situby the reaction of a hydrogen peroxide source and bleach activator, incombination with a bleach catalyst. Preferred bleach components comprisebleach catalysts, preferably organic bleach catalysts, as describedabove.

Particularly preferred bleach components are the bleach catalysts inparticular the organic bleach catalysts.

Fabric Hueinq Agents—

The composition may comprise a fabric hueing agent. Suitable fabrichueing agents include dyes, dye-clay conjugates, and pigments. Suitabledyes include small molecule dyes and polymeric dyes. Suitable smallmolecule dyes include small molecule dyes selected from the groupconsisting of dyes falling into the Color Index (C.I.) classificationsof Direct Blue, Direct Red, Direct Violet, Acid Blue, Acid Red, AcidViolet, Basic Blue, Basic Violet and Basic Red, or mixtures thereof.

In another aspect, suitable small molecule dyes include small moleculedyes selected from the group consisting of Color Index (Society of Dyersand Colorists, Bradford, UK) numbers Direct Violet 9, Direct Violet 35,Direct Violet 48, Direct Violet 51, Direct Violet 66, Direct Violet 99,Direct Blue 1, Direct Blue 71, Direct Blue 80, Direct Blue 279, Acid Red17, Acid Red 73, Acid Red 88, Acid Red 150, Acid Violet 15, Acid Violet17, Acid Violet 24, Acid Violet 43, Acid Red 52, Acid Violet 49, AcidViolet 50, Acid Blue 15, Acid Blue 17, Acid Blue 25, Acid Blue 29, AcidBlue 40, Acid Blue 45, Acid Blue 75, Acid Blue 80, Acid Blue 83, AcidBlue 90 and Acid Blue 113, Acid Black 1, Basic Violet 1, Basic Violet 3,Basic Violet 4, Basic Violet 10, Basic Violet 35, Basic Blue 3, BasicBlue 16, Basic Blue 22, Basic Blue 47, Basic Blue 66, Basic Blue 75,Basic Blue 159 and mixtures thereof. In another aspect, suitable smallmolecule dyes include small molecule dyes selected from the groupconsisting of Color Index (Society of Dyers and Colorists, Bradford, UK)numbers Acid Violet 17, Acid Violet 43, Acid Red 52, Acid Red 73, AcidRed 88, Acid Red 150, Acid Blue 25, Acid Blue 29, Acid Blue 45, AcidBlue 113, Acid Black 1, Direct Blue 1, Direct Blue 71, Direct Violet 51and mixtures thereof. In another aspect, suitable small molecule dyesinclude small molecule dyes selected from the group consisting of ColorIndex (Society of Dyers and Colorists, Bradford, UK) numbers Acid Violet17, Direct Blue 71, Direct Violet 51, Direct Blue 1, Acid Red 88, AcidRed 150, Acid Blue 29, Acid Blue 113 or mixtures thereof.

Suitable polymeric dyes include polymeric dyes selected from the groupconsisting of polymers containing conjugated chromogens (dye-polymerconjugates) and polymers with chromogens co-polymerized into thebackbone of the polymer and mixtures thereof.

In another aspect, suitable polymeric dyes include polymeric dyesselected from the group consisting of fabric-substantive colorants soldunder the name of Liquitint® (Milliken), dye-polymer conjugates formedfrom at least one reactive dye and a polymer selected from the groupconsisting of polymers comprising a moiety selected from the groupconsisting of a hydroxyl moiety, a primary amine moiety, a secondaryamine moiety, a thiol moiety and mixtures thereof. In still anotheraspect, suitable polymeric dyes include polymeric dyes selected from thegroup consisting of Liquitint® Violet CT, carboxymethyl cellulose (CMC)conjugated with a reactive blue, reactive violet or reactive red dyesuch as CMC conjugated with C.I. Reactive Blue 19, sold by Megazyme,Wicklow, Ireland under the product name AZO-CM-CELLULOSE, product codeS-ACMC, alkoxylated triphenyl-methane polymeric colorants, alkoxylatedthiophene polymeric colorants, and mixtures thereof.

Preferred hueing dyes include the whitening agents found in WO08/87497.These whitening agents may be characterized by the following structure(I):

wherein R₁ and R₂ can independently be selected from:

-   -   a) [(CH₂CR′HO)_(x)(CH₂CR″HO)_(y)H]

wherein R′ is selected from the group consisting of H, CH₃,CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof; wherein R″ is selected fromthe group consisting of H, CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof;wherein x+y≦5; wherein y≧1; and wherein z=0 to 5;

-   -   b) R₁=alkyl, aryl or aryl alkyl and        R₂=[(CH₂CR′HO)_(x)(CH₂CR″HO)_(y)H]

wherein R′ is selected from the group consisting of H, CH₃,CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof; wherein R″ is selected fromthe group consisting of H, CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof;wherein x+y≦10; wherein y≧1; and wherein z=0 to 5;

-   -   c) R₁=[CH₂CH₂(OR₃)CH₂OR₄] and R₂=[CH₂CH₂(OR₃)CH₂O R₄]

wherein R₃ is selected from the group consisting of H, (CH₂CH₂O)_(z)H,and mixtures thereof; and wherein z=0 to 10;

wherein R₄ is selected from the group consisting of (C₁-C₁₆)alkyl, arylgroups, and mixtures thereof; and

-   -   d) wherein R1 and R2 can independently be selected from the        amino addition product of styrene oxide, glycidyl methyl ether,        isobutyl glycidyl ether, isopropylglycidyl ether, t-butyl        glycidyl ether, 2-ethylhexylgycidyl ether, and glycidylhexadecyl        ether, followed by the addition of from 1 to 10 alkylene oxide        units.

A preferred whitening agent of the present invention may becharacterized by the following structure (II):

wherein R′ is selected from the group consisting of H, CH₃,CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof; wherein R″ is selected fromthe group consisting of H, CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof;wherein x+y≦5; wherein y≧1; and wherein z=0 to 5.

A further preferred whitening agent of the present invention may becharacterized by the following structure (Ill):

typically comprising a mixture having a total of 5 EO groups. Suitablepreferred molecules are those in Structure I having the followingpendant groups in “part a” above.

TABLE 1 R1 R2 R′ R″ x y R′ R″ x y a H H 3 1 H H 0 1 b H H 2 1 H H 1 1 c= b H H 1 1 H H 2 1 d = a H H 0 1 H H 3 1

Further whitening agents of use include those described in US2008134511(Unilever). A preferred agent is “Violet 13”.

Suitable dye clay conjugates include dye clay conjugates selected fromthe group comprising at least one cationic/basic dye and a smectiteclay, and mixtures thereof. In another aspect, suitable dye clayconjugates include dye clay conjugates selected from the groupconsisting of one cationic/basic dye selected from the group consistingof C.I. Basic Yellow 1 through 108, C.I. Basic Orange 1 through 69, C.I.Basic Red 1 through 118, C.I. Basic Violet 1 through 51, C.I. Basic Blue1 through 164, C.I. Basic Green 1 through 14, C.I. Basic Brown 1 through23, CI Basic Black 1 through 11, and a clay selected from the groupconsisting of Montmorillonite clay, Hectorite clay, Saponite clay andmixtures thereof. In still another aspect, suitable dye clay conjugatesinclude dye clay conjugates selected from the group consisting of:Montmorillonite Basic Blue B7 C.I. 42595 conjugate, MontmorilloniteBasic Blue B9 C.I. 52015 conjugate, Montmorillonite Basic Violet V3 C.I.42555 conjugate, Montmorillonite Basic Green G1 C.I. 42040 conjugate,Montmorillonite Basic Red R1 C.I. 45160 conjugate, Montmorillonite C.I.Basic Black 2 conjugate, Hectorite Basic Blue B7 C.I. 42595 conjugate,Hectorite Basic Blue B9 C.I. 52015 conjugate, Hectorite Basic Violet V3C.I. 42555 conjugate, Hectorite Basic Green G1 C.I. 42040 conjugate,Hectorite Basic Red R1 C.I. 45160 conjugate, Hectorite C.I. Basic Black2 conjugate, Saponite Basic Blue B7 C.I. 42595 conjugate, Saponite BasicBlue B9 C.I. 52015 conjugate, Saponite Basic Violet V3 C.I. 42555conjugate, Saponite Basic Green G1 C.I. 42040 conjugate, Saponite BasicRed R1 C.I. 45160 conjugate, Saponite C.I. Basic Black 2 conjugate andmixtures thereof.

Suitable pigments include pigments selected from the group consisting offlavanthrone, indanthrone, chlorinated indanthrone containing from 1 to4 chlorine atoms, pyranthrone, dichloropyranthrone,monobromodichloropyranthrone, dibromodichloropyranthrone,tetrabromopyranthrone, perylene-3,4,9,10-tetracarboxylic acid diimide,wherein the imide groups may be unsubstituted or substituted byC1-C3-alkyl or a phenyl or heterocyclic radical, and wherein the phenyland heterocyclic radicals may additionally carry substituents which donot confer solubility in water, anthrapyrimidinecarboxylic acid amides,violanthrone, isoviolanthrone, dioxazine pigments, copper phthalocyaninewhich may contain up to 2 chlorine atoms per molecule, polychloro-copperphthalocyanine or polybromochloro-copper phthalocyanine containing up to14 bromine atoms per molecule and mixtures thereof.

In another aspect, suitable pigments include pigments selected from thegroup consisting of Ultramarine Blue (CA. Pigment Blue 29), UltramarineViolet (CA. Pigment Violet 15) and mixtures thereof.

The aforementioned fabric hueing agents can be used in combination (anymixture of fabric hueing agents can be used). Suitable hueing agents aredescribed in more detail in U.S. Pat. No. 7,208,459. Preferred levels ofdye in compositions of the invention are 0.00001 to 0.5 wt %, or 0.0001to 0.25 wt %. The concentration of dyes preferred in water for thetreatment and/or cleaning step is from 1 ppb to 5 ppm, 10 ppb to 5 ppmor 20 ppb to 5 ppm. In preferred compositions, the concentration ofsurfactant will be from 0.2 to 3 g/l.

Encapsulates—

The composition may comprise an encapsulate. In one aspect, anencapsulate comprising a core, a shell having an inner and outersurface, said shell encapsulating said core.

In one aspect of said encapsulate, said core may comprise a materialselected from the group consisting of perfumes; brighteners; dyes;insect repellents; silicones; waxes; flavors; vitamins; fabric softeningagents; skin care agents in one aspect, paraffins; enzymes;anti-bacterial agents; bleaches; sensates; and mixtures thereof; andsaid shell may comprise a material selected from the group consisting ofpolyethylenes; polyamides; polyvinylalcohols, optionally containingother co-monomers; polystyrenes; polyisoprenes; polycarbonates;polyesters; polyacrylates; aminoplasts, in one aspect said aminoplastmay comprise a polyureas, polyurethane, and/or polyureaurethane, in oneaspect said polyurea may comprise polyoxymethyleneurea and/or melamineformaldehyde; polyolefins; polysaccharides, in one aspect saidpolysaccharide may comprise alginate and/or chitosan; gelatin; shellac;epoxy resins; vinyl polymers; water insoluble inorganics; silicone; andmixtures thereof.

In one aspect of said encapsulate, said core may comprise perfume.

In one aspect of said encapsulate, said shell may comprise melamineformaldehyde and/or cross linked melamine formaldehyde.

In a one aspect, suitable encapsulates may comprise a core material anda shell, said shell at least partially surrounding said core material,is disclosed. At least 75%, 85% or 90% of said encapsulates may have afracture strength of from 0.2 to 10 MPa, from 0.4 to 5 MPa, from 0.6 to3.5 MPa, or from 0.7 to 3 MPa; and a benefit agent leakage of from 0 to30%, from 0 to 20%, or from 0 to 5%.

In one aspect, at least 75%, 85% or 90% of said encapsulates may have aparticle size from 1 to 80 microns, from 5 to 60 microns, from 10 to 50microns, or from 15 to 40 microns.

In one aspect, at least 75%, 85% or 90% of said encapsulates may have aparticle wall thickness from 30 to 250 nm, from 80 to 180 nm, or from100 to 160 nm.

In one aspect, said encapsulates' core material may comprise a materialselected from the group consisting of a perfume raw material and/oroptionally a material selected from the group consisting of vegetableoil, including neat and/or blended vegetable oils including castor oil,coconut oil, cottonseed oil, grape oil, rapeseed, soybean oil, corn oil,palm oil, linseed oil, safflower oil, olive oil, peanut oil, coconutoil, palm kernel oil, castor oil, lemon oil and mixtures thereof; estersof vegetable oils, esters, including dibutyl adipate, dibutyl phthalate,butyl benzyl adipate, benzyl octyl adipate, tricresyl phosphate,trioctyl phosphate and mixtures thereof; straight or branched chainhydrocarbons, including those straight or branched chain hydrocarbonshaving a boiling point of greater than about 80° C.; partiallyhydrogenated terphenyls, dialkyl phthalates, alkyl biphenyls, includingmonoisopropylbiphenyl, alkylated naphthalene, includingdipropylnaphthalene, petroleum spirits, including kerosene, mineral oiland mixtures thereof; aromatic solvents, including benzene, toluene andmixtures thereof; silicone oils; and mixtures thereof.

In one aspect, said encapsulates' wall material may comprise a suitableresin including the reaction product of an aldehyde and an amine,suitable aldehydes include, formaldehyde. Suitable amines includemelamine, urea, benzoguanamine, glycoluril, and mixtures thereof.Suitable melamines include methylol melamine, methylated methylolmelamine, imino melamine and mixtures thereof. Suitable ureas includedimethylol urea, methylated dimethylol urea, urea-resorcinol, andmixtures thereof.

In one aspect, suitable formaldehyde scavengers may be employed with theencapsulates e.g. in a capsule slurry and/or added to a compositionbefore, during or after the encapsulates are added to such composition.Suitable capsules may be made by the following teaching ofUS2008/0305982; and/or US2009/0247449.

In a preferred aspect the composition can also comprise a depositionaid, preferably consisting of the group comprising cationic or nonionicpolymers. Suitable polymers include cationic starches, cationichydroxyethylcellulose, polyvinylformaldehyde, locust bean gum, mannans,xyloglucans, tamarind gum, polyethyleneterephthalate and polymerscontaining dimethylaminoethyl methacrylate, optionally with one ormonomers selected from the group comprising acrylic acid and acrylamide.

Perfumes—

In one aspect the composition comprises a perfume that comprises one ormore perfume raw materials selected from the group consisting of1,1′-oxybis-2-propanol; 1,4-cyclohexanedicarboxylic acid, diethyl ester;(ethoxymethoxy)cyclododecane; 1,3-nonanediol, monoacetate;(3-methylbutoxy)acetic acid, 2-propenyl ester; beta-methylcyclododecaneethanol;2-methyl-3-[(1,7,7-trimethylbicyclo[2.2.1]hept-2-yl)oxy]-1-propanol;oxacyclohexadecan-2-one; alpha-methyl-benzenemethanol acetate;trans-3-ethoxy-1,1,5-trimethylcyclohexane;4-(1,1-dimethylethyl)cyclohexanol acetate;dodecahydro-3a,6,6,9a-tetramethylnaphtho[2,1-b]furan; beta-methylbenzenepropanal; beta-methyl-3-(1-methylethyl)benzenepropanal;4-phenyl-2-butanone; 2-methylbutanoic acid, ethyl ester; benzaldehyde;2-methylbutanoic acid, 1-methylethyl ester;dihydro-5-pentyl-2(3H)furanone;(2E)-1-(2,6,6-trimethyl-2-cyclohexen-1-yl)-2-buten-1-one; dodecanal;undecanal; 2-ethyl-alpha, alpha-dimethylbenzenepropanal; decanal; alpha,alpha-dimethylbenzeneethanol acetate; 2-(phenylmethylene)octanal;2-[[3-[4-(1,1-dimethylethyl)phenyl]-2-methylpropylidene]amino]benzoicacid, methyl ester; 1-(2,6,6-trimethyl-3-cyclohexen-1-yl)-2-buten-1-one;2-pentylcyclopentanone; 3-oxo-2-pentyl cyclopentaneacetic acid, methylester; 4-hydroxy-3-methoxybenzaldehyde; 3-ethoxy-4-hydroxybenzaldehyde;2-heptylcyclopentanone; 1-(4-methylphenyl)ethanone;(3E)-4-(2,6,6-trimethyl-1-cyclohexen-1-yl)-3-buten-2-one;(3E)-4-(2,6,6-trimethyl-2-cyclohexen-1-yl)-3-buten-2-one;benzeneethanol; 2H-1-benzopyran-2-one; 4-methoxybenzaldehyde;10-undecenal; propanoic acid, phenylmethyl ester;beta-methylbenzenepentanol; 1,1-diethoxy-3,7-dimethyl-2,6-octadiene;alpha, alpha-dimethylbenzeneethanol;(2E)-1-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2-buten-1-one; acetic acid,phenylmethyl ester; cyclohexanepropanoic acid, 2-propenyl ester;hexanoic acid, 2-propenyl ester; 1,2-dimethoxy-4-(2-propenyl)benzene;1,5-dimethyl-bicyclo[3.2.1]octan-8-one oxime;4-(4-hydroxy-4-methylpentyl)-3-cyclohexene-1-carboxaldehyde;3-buten-2-ol; 2-[[[2,4(or3,5)-dimethyl-3-cyclohexen-1-yl]methylene]amino]benzoic acid, methylester; 8-cyclohexadecen-1-one; methyl ionone; 2,6-dimethyl-7-octen-2-ol;2-methoxy-4-(2-propenyl)phenol; (2E)-3,7-dimethyl-2,6-Octadien-1-ol;2-hydroxy-Benzoic acid, (3Z)-3-hexenyl ester; 2-tridecenenitrile;4-(2,2-dimethyl-6-methylenecyclohexyl)-3-methyl-3-buten-2-one;tetrahydro-4-methyl-2-(2-methyl-1-propenyl)-2H-pyran; Acetic acid,(2-methylbutoxy)-, 2-propenyl ester; Benzoic acid, 2-hydroxy-,3-methylbutyl ester; 2-Buten-1-one,1-(2,6,6-trimethyl-1-cyclohexen-1-yl)-, (Z)-; Cyclopentanecarboxylicacid, 2-hexyl-3-oxo-, methyl ester; Benzenepropanal, 4-ethyl-.alpha.,.alpha.-dimethyl-; 3-Cyclohexene-1-carboxaldehyde,3-(4-hydroxy-4-methylpentyl)-; Ethanone,1-(2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-1H-3a,7-methanoazulen-5-yl)-,[3R-(3.alpha.,3a.beta.,7.beta.,8a.alpha.)]-; Undecanal,2-methyl-2H-Pyran-2-one, 6-butyltetrahydro-; Benzenepropanal,4-(1,1-dimethylethyl)-.alpha.-methyl-; 2(3H)-Furanone, 5-heptyldihydro-;Benzoic acid, 2-[(7-hydroxy-3,7-dimethyloctylidene)amino]-, methyl;Benzoic acid, 2-hydroxy-, phenylmethyl ester; Naphthalene, 2-methoxy-;2-Cyclopenten-1-one, 2-hexyl-; 2(3H)-Furanone, 5-hexyldihydro-;Oxiranecarboxylic acid, 3-methyl-3-phenyl-, ethyl ester;2-Oxabicyclo[2.2.2]octane, 1,3,3-trimethyl-; Benzenepentanol,.gamma.-methyl-; 3-Octanol, 3,7-dimethyl-;3,7-dimethyl-2,6-octadienenitrile; 3,7-dimethyl-6-octen-1-ol; Terpineolacetate; 2-methyl-6-methylene-7-Octen-2-ol, dihydro derivative;3a,4,5,6,7,7a-hexahydro-4,7-Methano-1H-inden-6-ol propanoate;3-methyl-2-buten-1-ol acetate; (Z)-3-Hexen-1-ol acetate;2-ethyl-4-(2,2,3-trimethyl-3-cyclopenten-1-yl)-2-buten-1-ol;4-(octahydro-4,7-methano-5H-inden-5-ylidene)-butanal;3-2,4-dimethyl-cyclohexene-1-carboxaldehyde;1-(1,2,3,4,5,6,7,8-octahydro-2,3,8,8-tetramethyl-2-naphthalenyl)-ethanone;2-hydroxy-benzoic acid, methyl ester; 2-hydroxy-benzoic acid, hexylester; 2-phenoxy-ethanol; 2-hydroxy-benzoic acid, pentyl ester;2,3-heptanedione; 2-hexen-1-ol; 6-Octen-2-ol, 2,6-dimethyl-; damascone(alpha, beta, gamma or delta or mixtures thereof),4,7-Methano-1H-inden-6-ol, 3a,4,5,6,7,7a-hexahydro-, acetate;9-Undecenal; 8-Undecenal; Isocyclocitral; Ethanone,1-(1,2,3,5,6,7,8,8a-octahydro-2,3,8,8-tetramethyl-2-naphthalenyl)-;3-Cyclohexene-1-carboxaldehyde, 3,5-dimethyl-;3-Cyclohexene-1-carboxaldehyde, 2,4-dimethyl-; 1,6-Octadien-3-ol,3,7-dimethyl-; 1,6-Octadien-3-ol, 3,7-dimethyl-, acetate; Lilial(p-t-Bucinal), and Cyclopentanone,2-[2-(4-methyl-3-cyclohexen-1-yl)propyl]- and1-methyl-4-(1-methylethenyl)cyclohexene and mixtures thereof.

In one aspect the composition may comprise an encapsulated perfumeparticle comprising either a water-soluble hydroxylic compound ormelamine-formaldehyde or modified polyvinyl alcohol. In one aspect theencapsulate comprises (a) an at least partially water-soluble solidmatrix comprising one or more water-soluble hydroxylic compounds,preferably starch; and (b) a perfume oil encapsulated by the solidmatrix.

In a further aspect the perfume may be pre-complexed with a polyamine,preferably a polyethylenimine so as to form a Schiff base.

Polymers—

The composition may comprise one or more polymers. Examples arecarboxymethylcellulose, poly(vinyl-pyrrolidone), poly (ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleic/acrylic acid copolymersand lauryl methacrylate/acrylic acid co-polymers.

The composition may comprise one or more amphiphilic cleaning polymerssuch as the compound having the following general structure:bis((C₂H₅O)(C₂H₄O)n)(CH₃)—N⁺—C_(x)H_(2x)—N⁺—(CH₃)-bis((C₂H₅O)(C₂H₄O)n),wherein n=from 20 to 30, and x=from 3 to 8, or sulphated or sulphonatedvariants thereof.

The composition may comprise amphiphilic alkoxylated grease cleaningpolymers which have balanced hydrophilic and hydrophobic properties suchthat they remove grease particles from fabrics and surfaces. Specificembodiments of the amphiphilic alkoxylated grease cleaning polymers ofthe present invention comprise a core structure and a plurality ofalkoxylate groups attached to that core structure. These may comprisealkoxylated polyalkylenimines, preferably having an inner polyethyleneoxide block and an outer polypropylene oxide block.

Alkoxylated polycarboxylates such as those prepared from polyacrylatesare useful herein to provide additional grease removal performance. Suchmaterials are described in WO91/08281 and PCT90/01815. Chemically, thesematerials comprise polyacrylates having one ethoxy side-chain per every7-8 acrylate units. The side-chains are of the formula —(CH₂CH₂O)_(m)(CH₂)_(n)CH₃ wherein m is 2-3 and n is 6-12. The side-chains areester-linked to the polyacrylate “backbone” to provide a “comb” polymertype structure. The molecular weight can vary, but is typically in therange of 2000 to 50,000. Such alkoxylated polycarboxylates can comprisefrom 0.05 wt % to 10 wt % of the compositions herein.

The isoprenoid-derived surfactants of the present invention, and theirmixtures with other cosurfactants and other adjunct ingredients, areparticularly suited to be used with an amphilic graft co-polymer,preferably the amphilic graft co-polymer comprises (i) polyethyeleneglycol backbone; and (ii) and at least one pendant moiety selected frompolyvinyl acetate, polyvinyl alcohol and mixtures thereof. A preferredamphilic graft co-polymer is Sokalan HP22, supplied from BASF. Suitablepolymers include random graft copolymers, preferably a polyvinyl acetategrafted polyethylene oxide copolymer having a polyethylene oxidebackbone and multiple polyvinyl acetate side chains. The molecularweight of the polyethylene oxide backbone is preferably 6000 and theweight ratio of the polyethylene oxide to polyvinyl acetate is 40 to 60and no more than 1 grafting point per 50 ethylene oxide units.

Carboxylate Polymer—

The composition of the present invention may also include one or morecarboxylate polymers such as a maleate/acrylate random copolymer orpolyacrylate homopolymer. In one aspect, the carboxylate polymer is apolyacrylate homopolymer having a molecular weight of from 4,000 to9,000 Da, or from 6,000 to 9,000 Da.

Soil Release Polymer—

The composition of the present invention may also include one or moresoil release polymers having a structure as defined by one of thefollowing structures (I), (II) or (III):

—[(OCHR¹—CHR²)_(a)—O—OC—Ar—CO—]_(d)  (I)

—[(OCHR³—CHR⁴)_(b)—O—OC-sAr—CO—]_(e)  (II)

—[(OCHR⁵—CHR⁶)_(c)—OR⁷]_(f)  (III)

wherein:

a, b and c are from 1 to 200;

d, e and f are from 1 to 50;

Ar is a 1,4-substituted phenylene;

sAr is 1,3-substituted phenylene substituted in position 5 with SO₃Me;

Me is Li, K, Mg/2, Ca/2, Al/3, ammonium, mono-, di-, tri-, ortetraalkylammonium wherein the alkyl groups are C₁-C₁₈ alkyl or C₂-C₁₀hydroxyalkyl, or mixtures thereof;

R¹, R², R³, R⁴, R⁵ and R⁶ are independently selected from H or C₁-C₁₈n-or iso-alkyl; and

R⁷ is a linear or branched C₁-C₁₈ alkyl, or a linear or branched C₂-C₃₀alkenyl, or a cycloalkyl group with 5 to 9 carbon atoms, or a C₈-C₃₀aryl group, or a C₆-C₃₀ arylalkyl group.

Suitable soil release polymers are polyester soil release polymers suchas Repel-o-tex polymers, including Repel-o-tex, SF-2 and SRP6 suppliedby Rhodia. Other suitable soil release polymers include Texcarepolymers, including Texcare SRA100, SRA300, SRN100, SRN170, SRN240,SRN300 and SRN325 supplied by Clariant. Other suitable soil releasepolymers are Marloquest polymers, such as Marloquest SL supplied bySasol.

Cellulosic Polymer—

The composition of the present invention may also include one or morecellulosic polymers including those selected from alkyl cellulose, alkylalkoxyalkyl cellulose, carboxyalkyl cellulose, alkyl carboxyalkylcellulose. In one aspect, the cellulosic polymers are selected from thegroup comprising carboxymethyl cellulose, methyl cellulose, methylhydroxyethyl cellulose, methyl carboxymethyl cellulose, and mixuresthereof. In one aspect, the carboxymethyl cellulose has a degree ofcarboxymethyl substitution from 0.5 to 0.9 and a molecular weight from100,000 to 300,000 Da.

Enzymes—

The composition may comprise one or more enzymes which provide cleaningperformance and/or fabric care benefits. Examples of suitable enzymesinclude, but are not limited to, hemicellulases, peroxidases, proteases,cellulases, xylanases, lipases, phospholipases, esterases, cutinases,pectinases, mannanases, pectate lyases, keratinases, reductases,oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases,tannases, pentosanases, malanases, R-glucanases, arabinosidases,hyaluronidase, chondroitinase, laccase, and amylases, or mixturesthereof. A typical combination is an enzyme cocktail that may comprisee.g. a protease and lipase in conjunction with amylase. When present ina composition, the aforementioned additional enzymes may be present atlevels from 0.00001 to 2 wt %, from 0.0001 to 1 wt % or from 0.001 to0.5 wt % enzyme protein by weight of the composition.

In one aspect preferred enzymes would include a protease. Suitableproteases include metalloproteases and serine proteases, includingneutral or alkaline microbial serine proteases, such as subtilisins(EC3.4.21.62). Suitable proteases include those of animal, vegetable ormicrobial origin. In one aspect, such suitable protease may be ofmicrobial origin. The suitable proteases include chemically orgenetically modified mutants of the aforementioned suitable proteases.In one aspect, the suitable protease may be a serine protease, such asan alkaline microbial protease or/and a trypsin-type protease. Examplesof suitable neutral or alkaline proteases include:

(a) subtilisins (EC3.4.21.62), including those derived from Bacillus,such as Bacillus lentus, B. alkalophilus, B. subtilis, B.amyloliquefaciens, Bacillus pumilus and Bacillus gibsonii described inU.S. Pat. No. 6,312,936; U.S. Pat. No. 5,679,630; U.S. Pat. No.4,760,025; U.S. Pat. No. 7,262,042 and WO09/021867.

(b) trypsin-type or chymotrypsin-type proteases, such as trypsin (e.g.,of porcine or bovine origin), including the Fusarium protease describedin WO89/06270 and the chymotrypsin proteases derived from Cellumonasdescribed in WO05/052161 and WO05/052146.

(c) metalloproteases, including those derived from Bacillusamyloliquefaciens described in WO07/044993.

Preferred proteases include those derived from Bacillus gibsonii orBacillus Lentus.

Suitable commercially available protease enzymes include those soldunder the trade names Alcalase®, Savinase®, Primase®, Durazym®,Polarzyme®, Kannase®, Liquanase®, Liquanase Ultra®, Savinase Ultra®,Ovozyme®, Neutrase®, Everlase® and Esperase® by Novozymes, those soldunder the tradename Maxatase®, Maxacal®, Maxapem®, Properase®,Purafect®, Purafect Prime®, Purafect Ox®, FN3®, FN4®, Excellase®,Purafect OXP®, Opticlean® and Optimase® by Danisco, those available fromHenkel/Kemira, namely BLAP (sequence shown in FIG. 29 of U.S. Pat. No.5,352,604 with the following mutations S99D+S101R+S103A+V104I+G159S,hereinafter referred to as BLAP), BLAP R(BLAP+S3T+V4I+V199M+V205I+L217D), BLAP X (BLAP+S3T+V4I+V205I) and BLAPF49 (BLAP+S3T+V4I+A194P+V199M+V205I+L217D)—all from Henkel/Kemira; andKAP (Bacillus alkalophilus subtilisin with mutations A230V+S256G+S259N)from Kao.

Suitable alpha-amylases include those of bacterial or fungal origin.Chemically or genetically modified mutants (variants) are included. Apreferred alkaline alpha-amylase is derived from a strain of Bacillus,such as Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillusstearothermophilus, Bacillus subtilis, or other Bacillus sp., such asBacillus sp. NCIB12289, NCIB12512, NCIB12513, DSM9375 (U.S. Pat. No.7,153,818) DSM12368, DSMZ no.12649, KSM AP1378 (WO97/00324), KSM K36 orKSM K38 (EP1022334). Preferred amylases include:

(a) the variants described in WO94/02597, WO94/18314, WO96/23874 andWO97/43424, especially the variants with substitutions in one or more ofthe following positions versus the enzyme listed as SEQ ID No. 2 inWO96/23874: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188, 190,197, 202, 208, 209, 243, 264, 304, 305, 391, 408, and 444.

(b) the variants described in U.S. Pat. No. 5,856,164, WO99/23211,WO96/23873, WO00/60060 & WO06/002643, especially the variants with oneor more substitutions in the following positions versus the AA560 enzymelisted as SEQ ID No. 12 in WO06/002643: 26, 30, 33, 82, 37, 106, 118,128, 133, 149, 150, 160, 178, 182, 186, 193, 203, 214, 231, 256, 257,258, 269, 270, 272, 283, 295, 296, 298, 299, 303, 304, 305, 311, 314,315, 318, 319, 339, 345, 361, 378, 383, 419, 421, 437, 441, 444, 445,446, 447, 450, 461, 471, 482, 484, preferably that also contain thedeletions of D183* and G184*.

(c) variants exhibiting at least 90% identity with SEQ ID No. 4 inWO06/002643, the wild-type enzyme from Bacillus SP722, especiallyvariants with deletions in the 183 and 184 positions and variantsdescribed in WO00/60060, which is incorporated herein by reference.

(d) variants exhibiting at least 95% identity with the wild-type enzymefrom Bacillus sp.707 (SEQ ID NO:7 in U.S. Pat. No. 6,093,562),especially those comprising one or more of the following mutations M202,M208, S255, R172, and/or M261. Preferably said amylase comprises one ormore of M202L, M202V, M2025, M202T, M202I, M202Q, M202W, S255N and/orR172Q. Particularly preferred are those comprising the mutations M202Lor M202T.

(e) variants described in WO09/149130, preferably those exhibiting atleast 90% identity with SEQ ID NO: 1 or SEQ ID NO:2 in WO09/149130, thewild-type enzyme from Geobacillus Stearophermophilus or a truncatedversion thereof.

Suitable commercially available alpha-amylases include DURAMYL®,LIQUEZYME®, TERMAMYL®, TERMAMYL ULTRA®, NATALASE®, SUPRAMYL®,STAINZYME®, STAINZYME PLUS®, FUNGAMYL® and BAN® (Novozymes), KEMZYM® AT9000 Biozym Biotech Trading GmbH Wehlistrasse 27b A-1200 Wien Austria,RAPIDASE®, PURASTAR®, ENZYSIZE®, OPTISIZE HT PLUS®, POWERASE® andPURASTAR OXAM® (Danisco) and KAM® (Kao). In one aspect, suitableamylases include NATALASE®, STAINZYME® and STAINZYME PLUS® and mixturesthereof.

In one aspect, such enzymes may be selected from the group consistingof: lipases, including “first cycle lipases” such as those described inU.S. Pat. No. 6,939,702 and US2009/0217464. In one aspect, the lipase isa first-wash lipase, preferably a variant of the wild-type lipase fromThermomyces lanuginosus comprising one or more of the T231R and N233Rmutations. The wild-type sequence is the 269 amino acids (amino acids23-291) of the Swissprot accession number Swiss-Prot 059952 (derivedfrom Thermomyces lanuginosus (Humicola lanuginosa)). Preferred lipaseswould include those sold under the tradenames Lipex® and Lipolex®.

In one aspect, other preferred enzymes include microbial-derivedendoglucanases exhibiting endo-beta-1,4-glucanase activity (EC3.2.1.4),including a bacterial polypeptide endogenous to a member of the genusBacillus which has a sequence of at least 90%, 94%, 97% or 99% identityto the amino acid sequence SEQ ID NO:2 in U.S. Pat. No. 7,141,403 andmixtures thereof. Suitable endoglucanases are sold under the tradenamesCelluclean® and Whitezyme® (Novozymes).

Other preferred enzymes include pectate lyases sold under the tradenamesPectawash®, Pectaway®, Xpect® and mannanases sold under the tradenamesMannaway® (Novozymes), and Purabrite® (Danisco).

Dye Transfer Inhibiting Agents—

The compositions of the present invention may also include one or moredye transfer inhibiting agents. Suitable polymeric dye transferinhibiting agents include, but are not limited to, polyvinylpyrrolidonepolymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidoneand N-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles ormixtures thereof. When present in a composition, the dye transferinhibiting agents may be present at levels from 0.0001% to 10%, from0.01% to 5% or from 0.1% to 3% by weight of the composition.

Brighteners—

The compositions of the present invention can also contain additionalcomponents that may tint articles being cleaned, such as fluorescentbrighteners.

The composition may comprise C.I. fluorescent brightener 260 inalpha-crystalline form having the following structure:

In one aspect, the brightener is a cold water soluble brightener, suchas the C.I. fluorescent brightener 260 in alpha-crystalline form. In oneaspect the brightener is predominantly in alpha-crystalline form, whichmeans that typically at least 50 wt %, at least 75 wt %, at least 90 wt%, at least 99 wt %, or even substantially all, of the C.I. fluorescentbrightener 260 is in alpha-crystalline form.

The brightener is typically in micronized particulate form, having aweight average primary particle size of from 3 to 30 micrometers, from 3micrometers to 20 micrometers, or from 3 to 10 micrometers.

The composition may comprise C.I. fluorescent brightener 260 inbeta-crystalline form, and the weight ratio of: (i) C.I. fluorescentbrightener 260 in alpha-crystalline form, to (ii) C.I. fluorescentbrightener 260 in beta-crystalline form may be at least 0.1, or at least0.6. BE680847 relates to a process for making C.I fluorescent brightener260 in alpha-crystalline form.

Commercial optical brighteners which may be useful in the presentinvention can be classified into subgroups, which include, but are notnecessarily limited to, derivatives of stilbene, pyrazoline, coumarin,carboxylic acid, methinecyanines, dibenzothiophene-5,5-dioxide, azoles,5- and 6-membered-ring heterocycles, and other miscellaneous agents.Examples of such brighteners are disclosed in “The Production andApplication of Fluorescent Brightening Agents”, M. Zahradnik, Publishedby John Wiley & Sons, New York (1982). Specific nonlimiting examples ofoptical brighteners which are useful in the present compositions arethose identified in U.S. Pat. No. 4,790,856 and U.S. Pat. No. 3,646,015.

A further suitable brightener has the structure below:

Suitable fluorescent brightener levels include lower levels of from 0.01wt %, from 0.05 wt %, from 0.1 wt % or from 0.2 wt % to upper levels of0.5 wt % or 0.75 wt %.

In one aspect the brightener may be loaded onto a clay to form aparticle. Silicate salts —The compositions of the present invention canalso contain silicate salts, such as sodium or potassium silicate. Thecomposition may comprise of from 0 wt % to less than 10 wt % silicatesalt, to 9 wt %, or to 8 wt %, or to 7 wt %, or to 6 wt %, or to 5 wt %,or to 4 wt %, or to 3 wt %, or even to 2 wt %, and from above 0 wt %, orfrom 0.5 wt %, or from 1 wt % silicate salt. A suitable silicate salt issodium silicate.

Dispersants—

The compositions of the present invention can also contain dispersants.Suitable water-soluble organic materials include the homo- orco-polymeric acids or their salts, in which the polycarboxylic acidcomprises at least two carboxyl radicals separated from each other bynot more than two carbon atoms.

Enzyme Stabilizers—

Enzymes for use in compositions can be stabilized by various techniques.The enzymes employed herein can be stabilized by the presence ofwater-soluble sources of calcium and/or magnesium ions in the finishedfabric and home care compositions that provide such ions to the enzymes.In case of aqueous compositions comprising protease, a reversibleprotease inhibitor, such as a boron compound including borate, 4-formylphenylboronic acid, phenylboronic acid and derivatives thereof, orcompounds such as calcium formate, sodium formate and 1,2-propane diolcan be added to further improve stability.

Solvents—

Suitable solvents include water and other solvents such as lipophilicfluids. Examples of suitable lipophilic fluids include siloxanes, othersilicones, hydrocarbons, glycol ethers, glycerine derivatives such asglycerine ethers, perfluorinated amines, perfluorinated andhydrofluoroether solvents, low-volatility nonfluorinated organicsolvents, diol solvents, other environmentally-friendly solvents andmixtures thereof.

Structurant/Thickeners—

Structured liquids can either be internally structured, whereby thestructure is formed by primary ingredients (e.g. surfactant material)and/or externally structured by providing a three dimensional matrixstructure using secondary ingredients (e.g. polymers, clay and/orsilicate material). The composition may comprise a structurant, from0.01 to 5 wt %, or from 0.1 to 2.0 wt %. The structurant is typicallyselected from the group consisting of diglycerides and triglycerides,ethylene glycol distearate, microcrystalline cellulose, cellulose-basedmaterials, microfiber cellulose, hydrophobically modifiedalkali-swellable emulsions such as Polygel W30 (3VSigma), biopolymers,xanthan gum, gellan gum, and mixtures thereof. A suitable structurantincludes hydrogenated castor oil, and non-ethoxylated derivativesthereof. A suitable structurant is disclosed in U.S. Pat. No. 6,855,680.Such structurants have a thread-like structuring system having a rangeof aspect ratios. Other suitable structurants and the processes formaking them are described in WO10/034736.

Conditioning Agents—

The composition of the present invention may include a high meltingpoint fatty compound. The high melting point fatty compound usefulherein has a melting point of 25° C. or higher, and is selected from thegroup consisting of fatty alcohols, fatty acids, fatty alcoholderivatives, fatty acid derivatives, and mixtures thereof. Suchcompounds of low melting point are not intended to be included in thissection. Non-limiting examples of the high melting point compounds arefound in International Cosmetic Ingredient Dictionary, Fifth Edition,1993, and CTFA Cosmetic Ingredient Handbook, Second Edition, 1992.

The high melting point fatty compound is included in the composition ata level of from 0.1 to 40 wt %, from 1 to 30 wt %, from 1.5 to 16 wt %,from 1.5 to 8 wt % in view of providing improved conditioning benefitssuch as slippery feel during the application to wet hair, softness andmoisturized feel on dry hair.

The compositions of the present invention may contain a cationicpolymer. Concentrations of the cationic polymer in the compositiontypically range from 0.05 to 3 wt %, from 0.075 to 2.0 wt %, or from 0.1to 1.0 wt %. Suitable cationic polymers will have cationic chargedensities of at least 0.5 meq/gm, at least 0.9 meq/gm, at least 1.2meq/gm, at least 1.5 meq/gm, or less than 7 meq/gm, and less than 5meq/gm, at the pH of intended use of the composition, which pH willgenerally range from pH 3 to pH 9, or between pH 4 and pH 8. Herein,“cationic charge density” of a polymer refers to the ratio of the numberof positive charges on the polymer to the molecular weight of thepolymer. The average molecular weight of such suitable cationic polymerswill generally be between 10,000 and 10 million, between 50,000 and 5million, or between 100,000 and 3 million.

Suitable cationic polymers for use in the compositions of the presentinvention contain cationic nitrogen-containing moieties such asquaternary ammonium or cationic protonated amino moieties. Any anioniccounterions can be used in association with the cationic polymers solong as the polymers remain soluble in water, in the composition, or ina coacervate phase of the composition, and so long as the counterionsare physically and chemically compatible with the essential componentsof the composition or do not otherwise unduly impair compositionperformance, stability or aesthetics. Nonlimiting examples of suchcounterions include halides (e.g., chloride, fluoride, bromide, iodide),sulfate and methylsulfate.

Nonlimiting examples of such polymers are described in the CTFA CosmeticIngredient Dictionary, 3rd edition, edited by Estrin, Crosley, andHaynes, (The Cosmetic, Toiletry, and Fragrance Association, Inc.,Washington, D.C. (1982)).

Other suitable cationic polymers for use in the composition includepolysaccharide polymers, cationic guar gum derivatives, quaternarynitrogen-containing cellulose ethers, synthetic polymers, copolymers ofetherified cellulose, guar and starch. When used, the cationic polymersherein are either soluble in the composition or are soluble in a complexcoacervate phase in the composition formed by the cationic polymer andthe anionic, amphoteric and/or zwitterionic surfactant componentdescribed hereinbefore. Complex coacervates of the cationic polymer canalso be formed with other charged materials in the composition. Suitablecationic polymers are described in U.S. Pat. No. 3,962,418; U.S. Pat.No. 3,958,581; and US2007/0207109.

The composition of the present invention may include a nonionic polymeras a conditioning agent. Polyalkylene glycols having a molecular weightof more than 1000 are useful herein. Useful are those having thefollowing general formula:

wherein R⁹⁵ is selected from the group consisting of H, methyl, andmixtures thereof. Conditioning agents, and in particular silicones, maybe included in the composition. The conditioning agents useful in thecompositions of the present invention typically comprise a waterinsoluble, water dispersible, non-volatile, liquid that formsemulsified, liquid particles. Suitable conditioning agents for use inthe composition are those conditioning agents characterized generally assilicones (e.g., silicone oils, cationic silicones, silicone gums, highrefractive silicones, and silicone resins), organic conditioning oils(e.g., hydrocarbon oils, polyolefins, and fatty esters) or combinationsthereof, or those conditioning agents which otherwise form liquid,dispersed particles in the aqueous surfactant matrix herein. Suchconditioning agents should be physically and chemically compatible withthe essential components of the composition, and should not otherwiseunduly impair composition stability, aesthetics or performance.

The concentration of the conditioning agent in the composition should besufficient to provide the desired conditioning benefits. Suchconcentration can vary with the conditioning agent, the conditioningperformance desired, the average size of the conditioning agentparticles, the type and concentration of other components, and otherlike factors.

The concentration of the silicone conditioning agent typically rangesfrom 0.01 to 10 wt %. Non-limiting examples of suitable siliconeconditioning agents, and optional suspending agents for the silicone,are described in U.S. Reissue Pat. No. 34,584; U.S. Pat. No. 5,104,646;U.S. Pat. No. 5,106,609; U.S. Pat. No. 4,152,416; U.S. Pat. No.2,826,551; U.S. Pat. No. 3,964,500; U.S. Pat. No. 4,364,837; U.S. Pat.No. 6,607,717; U.S. Pat. No. 6,482,969; U.S. Pat. No. 5,807,956; U.S.Pat. No. 5,981,681; U.S. Pat. No. 6,207,782; U.S. Pat. No. 7,465,439;U.S. Pat. No. 7,041,767; U.S. Pat. No. 7,217,777; US2007/0286837A1;US2005/0048549A1; US2007/0041929A1; GB849433; DE10036533, which are allincorporated herein by reference; Chemistry and Technology of Silicones,N.Y.: Academic Press (1968); General Electric Silicone Rubber ProductData Sheets SE 30, SE 33, SE 54 and SE 76; Silicon Compounds, PetrarchSystems, Inc. (1984); and in Encyclopedia of Polymer Science andEngineering, vol. 15, 2d ed., pp 204-308, John Wiley & Sons, Inc.(1989).

The compositions of the present invention may also comprise from 0.05 to3 wt % of at least one organic conditioning oil as the conditioningagent, either alone or in combination with other conditioning agents,such as the silicones (described herein). Suitable conditioning oilsinclude hydrocarbon oils, polyolefins, and fatty esters. Also suitablefor use in the compositions herein are the conditioning agents describedin U.S. Pat. No. 5,674,478 and U.S. Pat. No. 5,750,122 or in U.S. Pat.No. 4,529,586; U.S. Pat. No. 4,507,280; U.S. Pat. No. 4,663,158; U.S.Pat. No. 4,197,865; U.S. Pat. No. 4,217,914; U.S. Pat. No. 4,381,919;and U.S. Pat. No. 4,422,853.

Hygiene and Malodour—

The compositions of the present invention may also comprise one or moreof zinc ricinoleate, thymol, quaternary ammonium salts such as Bardac®,polyethylenimines (such as Lupasol® from BASF) and zinc complexesthereof, silver and silver compounds, especially those designed toslowly release Ag⁺ or nano-silver dispersions.

Probiotics—

The compositions may comprise probiotics such as those described inWO09/043709.

Suds Boosters—

If high sudsing is desired, suds boosters such as the C₁₀-C₁₆alkanolamides or C₁₀-C₁₄ alkyl sulphates can be incorporated into thecompositions, typically at 1 to 10 wt % levels. The C₁₀-C₁₄ monoethanoland diethanol amides illustrate a typical class of such suds boosters.Use of such suds boosters with high sudsing adjunct surfactants such asthe amine oxides, betaines and sultaines noted above is alsoadvantageous. If desired, water-soluble magnesium and/or calcium saltssuch as MgCl₂, MgSO₄, CaCl₂, CaSO₄ and the like, can be added at levelsof, typically, 0.1 to 2 wt %, to provide additional suds and to enhancegrease removal performance.

Suds Suppressors—

Compounds for reducing or suppressing the formation of suds can beincorporated into the compositions of the present invention. Sudssuppression can be of particular importance in the so-called “highconcentration cleaning process” as described in U.S. Pat. No. 4,489,455and U.S. Pat. No. 4,489,574, and in front-loading-style washingmachines. A wide variety of materials may be used as suds suppressors,and suds suppressors are well known to those skilled in the art. Seee.g. Kirk Othmer Encyclopedia of Chemical Technology, Third Edition,Volume 7, pages 430-447 (John Wiley & Sons, Inc., 1979). Examples ofsuds supressors include monocarboxylic fatty acid and soluble saltstherein, high molecular weight hydrocarbons such as paraffin, fatty acidesters (e.g., fatty acid triglycerides), fatty acid esters of monovalentalcohols, aliphatic C₁₈-C₄₀ ketones (e.g., stearone), N-alkylated aminotriazines, waxy hydrocarbons preferably having a melting point belowabout 100° C., silicone suds suppressors, and secondary alcohols. Sudssupressors are described in U.S. Pat. No. 2,954,347; U.S. Pat. No.4,265,779; U.S. Pat. No. 4,265,779; U.S. Pat. No. 3,455,839; U.S. Pat.No. 3,933,672; U.S. Pat. No. 4,652,392; U.S. Pat. No. 4,978,471; U.S.Pat. No. 4,983,316; U.S. Pat. No. 5,288,431; U.S. Pat. No. 4,639,489;U.S. Pat. No. 4,749,740; U.S. Pat. No. 4,798,679; U.S. Pat. No.4,075,118; EP89307851.9; EP150872; and DOS 2,124,526.

For any detergent compositions to be used in automatic laundry washingmachines, suds should not form to the extent that they overflow thewashing machine. Suds suppressors, when utilized, are preferably presentin a “suds suppressing amount. By “suds suppressing amount” is meantthat the formulator of the composition can select an amount of this sudscontrolling agent that will sufficiently control the suds to result in alow-sudsing laundry detergent for use in automatic laundry washingmachines.

The compositions herein will generally comprise from 0 to 10 wt % ofsuds suppressor. When utilized as suds suppressors, monocarboxylic fattyacids, and salts therein, will be present typically in amounts up to 5wt %. Preferably, from 0.5 to 3 wt % of fatty monocarboxylate sudssuppressor is utilized. Silicone suds suppressors are typically utilizedin amounts up to 2.0 wt %, although higher amounts may be used.Monostearyl phosphate suds suppressors are generally utilized in amountsranging from 0.1 to 2 wt %. Hydrocarbon suds suppressors are typicallyutilized in amounts ranging from 0.01 to 5.0 wt %, although higherlevels can be used. The alcohol suds suppressors are typically used at0.2 to 3 wt %.

The compositions herein may have a cleaning activity over a broad rangeof pH. In certain embodiments the compositions have cleaning activityfrom pH 4 to pH 11.5. In other embodiments, the compositions are activefrom pH 8 to pH 10.

The compositions herein may have cleaning activity over a wide range oftemperatures, e.g., from 10° C. or lower to 90° C. Preferably thetemperature will be below 50° C. or 40° C. or even 30° C. In certainembodiments, the optimum temperature range for the compositions is from10° C. to 20° C., from 15° C. to 25° C., from 15° C. to 30° C., from 20°C. to 30° C., from 25° C. to 35° C., from 30° C. to 40° C., from 35° C.to 45° C., or from 40° C. to 50° C.

The compositions herein may further be stabilized by the addition ofdivalent cations such as calcium (Ca²⁺), magnesium (Mg²⁺) and iron-II(Fe²⁺) or any combination thereof.

In some aspects the invention relates to a composition, wherein thecomposition is more effective in hydrolyzing lipid substrates comparedto an equivalent composition comprising Thermomyces lanuginosus lipase(Lipolase™) in place of Lipr139 lipase. In other aspects the compositioncomprises Lipex™ in place of Lipr139 lipase.

Form of the Composition

The cleaning compositions described herein are advantageously employedfor example, in laundry applications, hard surface cleaning, dishwashingapplications, as well as cosmetic applications such as dentures, teeth,hair and skin. In addition, due to the unique advantages of increasedeffectiveness in lower temperature solutions, the Lipr139 enzymedescribed herein are ideally suited for laundry applications. Thecompositions of the invention are in particular solid or liquid cleaningand/or treatment compositions. In one aspect the invention relates to acleaning composition, wherein the form of the composition is selectedfrom the group consisting of a liquid, a gel, a soap bar, a powder, agranulated solid, a tablet, or any combination thereof. In anotheraspect of the invention, the composition has a single or amulti-compartment unit dose form.

Water-Soluble Film—

The compositions of the present invention may also be encapsulatedwithin a water-soluble film. Preferred film materials are preferablypolymeric materials. The film material can e.g. be obtained by casting,blow-moulding, extrusion or blown extrusion of the polymeric material,as known in the art. Preferred polymers, copolymers or derivativesthereof suitable for use as pouch material are selected from polyvinylalcohols, polyvinyl pyrrolidone, polyalkylene oxides, acrylamide,acrylic acid, cellulose, cellulose ethers, cellulose esters, celluloseamides, polyvinyl acetates, polycarboxylic acids and salts,polyaminoacids or peptides, polyamides, polyacrylamide, copolymers ofmaleic/acrylic acids, polysaccharides including starch and gelatine,natural gums such as xanthum and carragum. More preferred polymers areselected from polyacrylates and water-soluble acrylate copolymers,methylcellulose, carboxymethylcellulose sodium, dextrin, ethylcellulose,hydroxyethyl cellulose, hydroxypropyl methylcellulose, maltodextrin,polymethacrylates, and most preferably selected from polyvinyl alcohols,polyvinyl alcohol copolymers and hydroxypropyl methyl cellulose (HPMC),and combinations thereof. Preferably, the level of polymer in the pouchmaterial, e.g. a PVA polymer, is at least 60 wt %. The polymer can haveany weight average molecular weight, preferably from about 1.000 to1.000.000, from about 10.000 to 300.000, from about 20.000 to 150.000.Mixtures of polymers can also be used as the pouch material.

Naturally, different film material and/or films of different thicknessmay be employed in making the compartments of the present invention. Abenefit in selecting different films is that the resulting compartmentsmay exhibit different solubility or release characteristics.

Preferred film materials are PVA films known under the MonoSol tradereference M8630, M8900, H8779 and those described in U.S. Pat. No.6,166,117 and U.S. Pat. No. 6,787,512 and PVA films of correspondingsolubility and deformability characteristics.

The film material herein can also comprise one or more additiveingredients. For example, it can be beneficial to add plasticisers, e.g.glycerol, ethylene glycol, diethyleneglycol, propylene glycol, sorbitoland mixtures thereof. Other additives include functional detergentadditives to be delivered to the wash water, e.g. organic polymericdispersants, etc.

Processes of Making the Compositions

The compositions of the present invention can be formulated into anysuitable form and prepared by any process chosen by the formulator,non-limiting examples of which are described in Applicants' examples andin U.S. Pat. No. 4,990,280; US20030087791A1; US20030087790A1;US20050003983A1; US20040048764A1; U.S. Pat. No. 4,762,636; U.S. Pat. No.6,291,412; US20050227891A1; EP1070115A2; U.S. Pat. No. 5,879,584; U.S.Pat. No. 5,691,297; U.S. Pat. No. 5,574,005; U.S. Pat. No. 5,569,645;U.S. Pat. No. 5,565,422; U.S. Pat. No. 5,516,448; U.S. Pat. No.5,489,392; U.S. Pat. No. 5,486,303 all of which are incorporated hereinby reference. The compositions of the invention or prepared according tothe invention comprise cleaning and/or treatment composition including,but not limited to, compositions for treating fabrics, hard surfaces andany other surfaces in the area of fabric and home care, including: aircare including air fresheners and scent delivery systems, car care,dishwashing, fabric conditioning (including softening and/orfreshening), laundry detergency, laundry and rinse additive and/or care,hard surface cleaning and/or treatment including floor and toilet bowlcleaners, granular or powder-form all-purpose or “heavy-duty” washingagents, especially cleaning detergents; liquid, gel or paste-formall-purpose washing agents, especially the so-called heavy-duty liquidtypes; liquid fine-fabric detergents; hand dishwashing agents or lightduty dishwashing agents, especially those of the high-foaming type;machine dishwashing agents, including the various tablet, granular,liquid and rinse-aid types for household and institutional use: car orcarpet shampoos, bathroom cleaners including toilet bowl cleaners; aswell as cleaning auxiliaries such as bleach additives and “stain-stick”or pre-treat types, substrate-laden compositions such as dryer addedsheets. Preferred are compositions and methods for cleaning and/ortreating textiles and/or hard surfaces, most preferably textiles. Thecompositions are preferably compositions used in a pre-treatment step ormain wash step of a washing process, most preferably for use in textilewashing step.

As used herein, the term “fabric and/or hard surface cleaning and/ortreatment composition” is a subset of cleaning and treatmentcompositions that includes, unless otherwise indicated, granular orpowder-form all-purpose or “heavy-duty” washing agents, especiallycleaning detergents; liquid, gel or paste-form all-purpose washingagents, especially the so-called heavy-duty liquid types; liquidfine-fabric detergents; hand dishwashing agents or light dutydishwashing agents, especially those of the high-foaming type; machinedishwashing agents, including the various tablet, granular, liquid andrinse-aid types for household and institutional use; liquid cleaning anddisinfecting agents, car or carpet shampoos, bathroom cleaners includingtoilet bowl cleaners; fabric conditioning compositions includingsoftening and/or freshening that may be in liquid, solid and/or dryersheet form; as well as cleaning auxiliaries such as bleach additives and“stain-stick” or pre-treat types, substrate-laden compositions such asdryer added sheets. All of such compositions which are applicable may bein standard, concentrated or even highly concentrated form even to theextent that such compositions may in certain aspect be non-aqueous.

Method of Use

The present invention includes a method for cleaning any surfaceincluding treating a textile or a hard surface or other surfaces in thefield of fabric and/or home care. In a preferred aspect of theinvention, the method comprises the step of contacting the surface to betreated in a pre-treatment step or main wash step of a washing process,most preferably for use in a textile washing step or alternatively foruse in dishwashing including both manual as well as automated/mechanicaldishwashing. In one embodiment of the invention the lipase variant andother components are added sequentially into the method for cleaningand/or treating the surface. Alternatively, the lipase variant and othercomponents are added simultaneously.

As used herein, washing includes but is not limited to, scrubbing, andmechanical agitation. Washing may be conducted with a foam compositionas described in WO08/101958 and/or by applying alternating pressure(pressure/vaccum) as an addition or as an alternative to scrubbing andmechanical agitation. Drying of such surfaces or fabrics may beaccomplished by any one of the common means employed either in domesticor industrial settings. The cleaning compositions of the presentinvention are ideally suited for use in laundry as well as dishwashingapplications. Accordingly, the present invention includes a method forcleaning an object including but not limiting to fabric, tableware,cutlery and kitchenware. The method comprises the steps of contactingthe object to be cleaned with a said cleaning composition comprising atleast one embodiment of Applicants' cleaning composition, cleaningadditive or mixture thereof. The fabric may comprise most any fabriccapable of being laundered in normal consumer or institutional useconditions. The solution may have a pH from 8 to 10.5. The compositionsmay be employed at concentrations from 500 to 15.000 ppm in solution.The water temperatures typically range from 5° C. to 90° C. The water tofabric ratio is typically from 1:1 to 30:1.

In one aspect the invention relates to a method of using Lipr139 forproducing a cleaning composition. In one aspect the invention relates touse of the cleaning composition for cleaning an object.

In one aspect the invention relates to a method of stabilizing acleaning composition, comprising adding a lipase with at least 75%identity to SEQ ID NO: 2 to the composition wherein the stability of thecomposition is greater than the stability of an equivalent compositioncomprising Thermomyces lanuginosus lipase in place of the lipase with atleast 75% identity to SEQ ID NO: 2, preferably wherein stability ismeasured in a final cleaning composition and/or in a final wash medium.

In one aspect the invention relates to a method of producing thecomposition, comprising adding a lipase with at least 75% identity toSEQ ID NO: 2, and a surfactant. In one aspect the invention relates to amethod for cleaning a surface, comprising contacting a lipid stainpresent on the surface to be cleaned with the cleaning composition. Inone aspect the invention relates to a method for hydrolyzing a lipidpresent in a soil and/or a stain on a surface, comprising contacting thesoil and/or the stain with the cleaning composition.

EXAMPLES

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

Media and Solutions

Unless otherwise indicated the materials are of reagent grade. Thecommercially available enzymes Lipolase™ and Lipex™ were obtained fromNovozymes A/S.

Example 1 Identification, Cloning and Expression

Chromosomal DNA from Cedecea sp-16640 was isolated by QIAamp DNA BloodMini Kit (Qiagen, Hilden, Germany). 5 ug of chromosomal DNA were sentfor Illumina sequencing at FASTERIS SA, Switzerland. The genomesequences were analyzed for open reading frames encoding lipolyticenzymes and the Lipr139 lipase was identified.

The nucleotide sequence of the Lipr139 synthetic gene set forth as SEQID NO: 1 was synthesized by Gene Art (GENEART AG BioPark,Josef-Engert-Str. 11, 93053, Regensburg, Germany). The gene sequenceencoding Lipr139 was verified by Sanger sequencing. The synthetic genewas subcloned using Sacl and Mlul restriction sites into Bacillusexpression vector as described in WO12/025577. Transformants wereselected on LB plates supplemented with 6 ug/ml chloramphenicol.

The recombinant Bacillus subtilis clone containing the integratedexpression construct was selected and cultivated on a rotary shakingtable in 500 ml baffled Erlenmeyer flasks each containing 10 mlcasein-based media supplemented with 34 mg/l chloramphenicol. The clonewas cultivated for 5 days at 26° C. The enzyme containing supernatantswere harvested and the enzyme purified as described below.

Example 2 Purification

The fermentation supernatant was filtered through PES Bottle top filterwith a 0.22 um cut-off (Cat.no. 567-0020, Thermo Fisher Scientific,Kastrupsvej 90, 4000 Roskilde, Denmark) adjusted to 1M NaCl and appliedto HIC/affinity chromatography on a 50 ml Decylamine agarose column(Acetyleret Decylaminagarose, Cat.no. CS76, UpFront Cromatography A/S,Lersø Parkalle 42, 2100 Copenhagen Ø, Denmark). The column was washedwith 3 column volumes (CV) Buffer A (50 mM Borate; 1M NaCl; pH 9) andthe protein was subsequently eluted with 3 CV 100% Buffer B (50 mMBorate; 30% isopropanol; pH 9). Elution of the protein was monitored at280 nm. Fractions with high 280 nm absorbance were analysed on SDS-Pageon their Lipr139 content.

Fractions of pure protein were pooled, diluted with MilliQ H₂O untilconductivity was below 3 mS/cm and then loaded onto a 50 ml Q-sepharosefast flow column (Cat. No. 17-0510-01 GE Healthcare Europe GmbH, ParkAllé 295, 2605 Brondby, Denmark). The column was washed with 3 CV BufferA (50 mM Borate; pH 9) and the protein was subsequently eluted withBuffer B (50 mM Borate; 1M NaCl; pH 9) on a gradient from 0%-100%.Elution of the protein was monitored at 280 nm and the majority of theprotein eluted at around 50% BufferB.

The fractions were analysed on SDS-Page and pure fractions were pooled.The purified protein had an estimated MW of 30 kD and a purity >95%.

The amino acid sequence of the mature Lipr139 lipase as set forth in SEQID NO: 2 was verified by In-gel digest.

Example 3 Hydrolytic Activity on Triglycerides

Plate Assay

The hydrolytic activity of the lipase on various substrates wasinvestigated using the following agar plates: Olive oil plates (1% Oliveoil; 1% Litex Agarose HSH 1000; 1 mM CaCl₂; 50 mM Hepes pH 8.0),Tributyrin plates (1.25% tributyrin; 1.65% gum Arabic; 1% Litex AgaroseHSH 1000; 50 mM Hepes; 0.008% brilliant green; pH 7.0) and Lecithinplates (1% Phosphatidylcholin; 1% Litex Agarose HSH 1000; 50 mM Acetate;1 mM CaCl₂; pH 5.5).

The hydrolytic activity at various pH was assessed on Olive oil plateswith the following pHs: pH 7, pH 8, pH 9 and pH 10 (1% Olive oil; 1%Litex Agarose HSH 1000; 1 mM CaCl₂; 50 mM Hepes (pH 7 & 8) or 50 mMBorate (pH 9 & 10)).

20 ul aliquots of the purified enzyme, buffer (negative control) andLipolase™ and Lipex™ (positive controls) were each distributed intopunched holes with a diameter of 3 mm and incubated for 24 hours at 20°C. The plates were subsequently examined for the presence or absence ofa clearing zone around the holes. Hydrolytic activity was indicated by aclearing zone around the holes. The negative control did not give riseto a clearing zone.

Lipr139 showed hydrolytic activity on all three substrates tested. OnOlive oil plates Lipr139 showed comparable hydrolytic activity withLipolase™ and a higher activity than Lipex™. On Tributyrin platesLipr139 showed a comparable activity with Lipolase™ and Lipex™. OnLecithin plates Lipr139 showed an increased activity in comparison withboth Lipolase™ and Lipex™.

Lipr139 showed hydrolytic activity at pH 8, pH 9 and pH 10 and maximumactivity at pH 9.

Example 4 Hydrolytic Activity on Fatty Acids

pNP Assay

The hydrolytic activity of Lipr139 on various fatty acids wasinvestigated in a kinetic assay using p-nitrophenyl acyl esters assubstrate. The 100 mM stock solutions in DMSO of the substrates:p-Nitrophenyl butyrate (C3), p-Nitrophenyl caproate (C6), p-Nitrophenylcaprate (C10), p-Nitrophenyl laurate (C12) and p-Nitrophenyl palmitate(C16) (all from Sigma-Aldrich Danmark A/S, Kirkebjerg Allé 84, 2605Brøndby; Cat.no.: 03:N-9876, C6: N-0502, 010: N-0252, C12: N-2002, C16:N-2752) were diluted to a final concentration of 1 mM into assay buffer(50 mM Tris; pH 7.7; 0.4% TritonX-100).

Lipr139 and the controls: Lipolase™ and Lipex™ (in 50 mM Hepes; pH 8.0;10 ppm TritonX-100; +/−20 mM CaCl₂) were added to the substrate solutionin the following final concentrations: 0.01 mg/ml; 5×10⁻³ mg/ml; 2.5×10⁴mg/ml; and 1.25×10⁴ mg/ml in 96-well NUNC plates (Cat. No:260836,Kamstrupvej 90, DK-4000, Roskilde). Release of p-nitrophenol byhydrolysis of p-nitrophenyl acyl was monitored at 405 nm for 5 minutesin 10 second intervals on a Spectra max 190 (Molecular Devices GmbH,Bismarckring 39, 88400 Biberach an der Riss, GERMANY).

The hydrolytic activity of Lipr139 for all substrates measured wassignificantly higher in the presence of 2 mM CaCl₂ in comparison withthe activity measured in the absence of CaCl₂

In the presence of CaCl₂ Lipr139 showed a lower (40-50%) hydrolyticactivity than Lipolase™ and Lipex™ for all chain-lengths tested. Themaximum activity for Lipr139 was measured for pNP-caprate (C10).

In the absence of 2 mM CaCl₂ the Lipr 139 showed its highest activitywith pNP-caprate (010) for which the activity was higher than thecontrols Lipex™ and Lipolase™. For the other substrates the activity wascomparable to Lipolase™ and Lipex™.

Example 5 Stability

The thermostability of the lipases was determined by DifferentialScanning calorimetry (DSC) using a VP Differential Scanning calorimeter(MicroCal Inc., Piscataway, N.J., USA). The thermal denaturationtemperature, Td (° C.), was taken as the top of denaturation peak (majorendothermic peak) in thermograms (Cp vs. T) obtained after heatingenzyme solutions in selected buffers at a constant programmed heatingrate of 90 K/hr. Sample- and reference-solutions (approximately 0.5 ml)were loaded into the calorimeter (reference: buffer without enzyme) andthermally pre-equilibrated for 10 minutes at 25° C. prior to DSC scanfrom 25° C. to 110° C. Denaturation temperatures were determined with anaccuracy of approximately +/−1° C.

At pH 8 (50 mM HEPES) the thermal denaturation temperature was measuredto 73° C.

Example 6 Relative Wash Performance

In order to assess the wash performance in laundry washing experimentswere performed, using the Automatic Mechanical Stress Assay (AMSA). TheAMSA plate has a number of slots for test solutions and a lid firmlysqueezing the laundry sample, the textile to be washed against all theslot openings. During the washing time, the plate, test solutions,textile and lid were vigorously shaken to bring the test solution incontact with the textile and apply mechanical stress in a regular,periodic oscillating manner. For further description see WO02/42740especially the paragraph “Special method embodiments” at page 23-24.

The laundry experiments were conducted in glycine buffers at differentpH and in Compositions with different surfactant level and withdifferent pH. The experimental conditions are specified below.

TABLE 2 Compositions/ 50 mM glycine buffer pH 8 buffers 50 mM glycinebuffer pH 9 50 mM glycine buffer pH 10 3.3 g/L Composition 0%surfactant, 50 mM glycine buffer pH 8 3.3 g/L Composition 0% surfactant,50 mM glycine buffer pH 9 3.3 g/L Composition 0% surfactant, 50 mMglycine buffer pH 10 3.3 g/L Composition 10% surfactant, 50 mM glycinebuffer pH 8 3.3 g/L Composition 10% surfactant, 50 mM glycine buffer pH9 3.3 g/L Composition 10% surfactant, 50 mM glycine buffer pH 10 3.3 g/LComposition 20% surfactant, 50 mM glycine buffer pH 8 3.3 g/LComposition 20% surfactant, 50 mM glycine buffer pH 9 3.3 g/LComposition 20% surfactant, 50 mM glycine buffer pH 10 3.3 g/LComposition 60% surfactant, 50 mM glycine buffer pH 8 3.3 g/LComposition 60% surfactant, 50 mM glycine buffer pH 9 3.3 g/LComposition 60% surfactant, 50 mM glycine buffer pH 10 3.3 g/LComposition 100% surfactant, 50 mM glycine buffer pH 8 Test solution 160microliter volume Wash time 15 minutes Temperature 25° C. Water hardness15° dH Lipase dosage 0 ppm & 1 ppm Test material Cream turmeric stainaccording to WO06/125437

TABLE 3 Total Composition (wt %) surfactant comprised 0% 10% 20% 60%100% linear 0 1.3 2.6 7.8 13 alkylbenzenesulfonic acid (LAS) (MarlonAS3) sodium alkyl(C12)ether 0 1 2 6 10 sulfate (AEOS) (STEOL CS-370 E)alcohol ethoxylate (AEO) 0 1.1 2.2 6.6 11 (Bio-Soft N25-7) coco soap(Radiacid 631) 2.75 2.75 2.75 2.75 2.75 soy soap (Edenor SJ) 2.75 2.752.75 2.75 2.75 sodium hydroxide 0 0.2 0.4 1.2 2 ethanol 3 3 3 3 3propane-1,2-diol (MPG) 6 6 6 6 6 glycerol 2 2 2 2 2 triethanolamine(TEA) 3 3 3 3 3 sodium formate 1 1 1 1 1 sodium citrate 2 2 2 2 2Diethylenetria- 0.2 0.2 0.2 0.2 0.2 minepentakis (methylenephosphonicacid) (DTMPA) polycarboxylate polymer 0.2 0.2 0.2 0.2 0.2 (PCA) (SokalanCP-5) Water up to 100 100 100 100 100

Final adjustments to the specified pH were done with NaOH or citricacid. Water hardness was adjusted to 15° dH by addition of CaCl₂ andMgCl₂ (Ca²⁺:Mg²⁺=4:1) to the test system.

After washing the textiles were flushed in tap water and excess waterwas removed from the textiles using filter paper and immediatelythereafter the textiles were dried at 85° C. for 5 min.

The wash performance was measured as the color change of the washedsoiled textile. The soil was cream mixed with turmeric. Turmericcontains the colorant curcumin, which function as a pH indicator byhaving pH dependent color change. Lipase activity leads to release offree fatty acids from the cream acylglycerides and this leads to pHdecrease and thereby color change of the curcumin pH indicator. Lipasewash performance can therefore be expressed as the extent of colorchange of light reflected-emitted from the washed soiled textile whenilluminated with white light.

Color measurements were made with a professional flatbed scanner (KodakiQsmart, Kodak, Midtager 29, DK-2605 Brøndby, Denmark), which was usedto capture an image of the washed soiled textile. To extract a value forthe light intensity from the scanned images, 24-bit pixel values fromthe image were converted into values for red, green and blue (RGB). Theintensity value (Int) was calculated by adding the RGB values togetheras vectors and then taking the length of the resulting vector:

Int=√{square root over (r ² +g ² +b ²)}

Color change due to lipase activity was measured as the increase in thereflection-emitting of green light (G) relative to the intensity (Int)of the total reflected-emitted light. The wash performance (RP(Wash)) ofa lipase relative to a reference lipase (Lipolase™) was calculated as:RP(Wash)=(G/Int(tested lipase)−G/Int(no enzyme))/(G/Int(lipaseref.)−G/Int(no enzyme)).

TABLE 4 shows the Relative wash performance, RP(Wash) of the testedlipase

Lipolase Lipr139 pH RP(wash) RP(wash) Buffer 0% surfactant 8 1.00 1.10 91.00 1.40 10 1.00 2.19 Composition 0% surfactant 8 1.00 1.18 9 1.00 1.8910 1.00 2.47 Composition 10% surfactant 8 1.00 1.80 9 1.00 2.89 10 1.002.76 Composition 20% surfactant 8 1.00 1.90 9 1.00 2.96 10 1.00 3.86Composition 60% surfactant 8 1.00 1.91 9 1.00 1.89 10 1.00 2.38Composition 100% surfactant 8 1.00 2.59

Furthermore, the relative wash performance (RP(Wash)) of Lipr139 was atlevel with the lipase Lipex™ when tested in Compositions containing10%-100% surfactant.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

1. An isolated polypeptide having lipase activity, selected from thegroup consisting of: (a) a polypeptide having at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% sequence identity to the mature polypeptideof SEQ ID NO: 2; (b) a polypeptide encoded by a polynucleotide thathybridizes under low stringency conditions, medium stringencyconditions, medium-high stringency conditions, high stringencyconditions, or very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 1, or (ii) the full-lengthcomplement of (i); (c) a polypeptide encoded by a polynucleotide havingat least 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO: 1; (d)a variant of the polypeptide of (a), (b) or (c) comprising asubstitution, deletion, and/or insertion at one or more positions; and(e) a fragment of the polypeptide of (a), (b), (c), or (d) that haslipase activity.
 2. The polypeptide of claim 1, comprising or consistingof SEQ ID NO: 2 or the mature polypeptide of SEQ ID NO:
 2. 3. Thepolypeptide of claim 2, wherein the mature polypeptide is amino acids 1to 292 of SEQ ID NO:
 2. 4. A composition comprising the polypeptide ofany of claims 1-3.
 5. The composition of claim 4, wherein saidcomposition is more effective in removing lipid stains present at asurface in comparison with an equivalent composition lacking the lipase.6. The composition of any of claims 4-5 further comprising one or moredivalent cations, preferably selected from calcium (Ca²⁺), magnesium(Mg²⁺) and iron-II (Fe²⁺).
 7. The composition of any of claims 4-6,further comprising one or more surfactants selected from the groupconsisting of an anionic surfactant, a cationic surfactant, a non-ionicsurfactant, zwitterionic surfactant.
 8. The composition of any of claims4-7, wherein the surfactant comprises one or more surfactants selectedfrom the group consisting of sodium dodecyl benzene sulfonate, sodiumhydrogenated cocoate, sodium laureth sulfate, C12-14 pareth-7, C12-15pareth-7, sodium C12-15 pareth sulfate, and C14-15 pareth-4.
 9. Thecomposition of any of claims 4-8, formulated at a pH from 8.0 to
 11. 10.The composition of any of claims 4-9, wherein the composition isselected from the group consisting of a laundry cleaning composition, adishwashing cleaning composition, a hard-surface cleaning compositionand a personal care cleaning composition.
 11. The composition of any ofclaims 4-10, wherein the form of the composition is selected from thegroup consisting of a liquid, a gel, a soap bar, a powder, a granulatedsolid, a tablet, or any combination thereof.
 12. The composition of anyof claims 4-11, wherein the composition is effective in hydrolyzing alipid at a temperature of from about 10° C. to 90° C.
 13. Thecomposition of any of claims 7-12, wherein the composition is moreeffective in hydrolyzing lipid substrates compared to an equivalentcomposition comprising Thermomyces lanuginosus lipase (Lipolase™) inplace of Lipr139 lipase.
 14. The composition of any of claims 4-13,further comprising one or more enzymes selected from: hemicellulases,peroxidases, proteases, cellulases, xylanases, lipases, phospholipases,esterases, cutinases, pectinases, mannanases, pectate lyases,keratinases, reductases, oxidases, phenoloxidases, lipoxygenases,ligninases, pullulanases, tannases, pentosanases, malanases,R-glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase,amylases, or mixtures thereof.
 15. The composition of claims 4-14,wherein the stability of the lipase with at least 92% identity to SEQ IDNO: 2 is greater than the stability of the Thermomyces lanuginosuslipase in an equivalent composition comprising Thermomyces lanuginosuslipase in place of the lipase with at least 92% identity to SEQ ID NO:2, preferably wherein stability is measured in a final compositionand/or in a final wash medium.
 16. A method of producing the compositionaccording to any of claims 4-15, comprising adding a lipase with atleast 92% identity to SEQ ID NO: 2, and a surfactant.
 17. A method forcleaning a surface, comprising contacting a lipid stain present on thesurface to be cleaned with the composition according to any of claims4-15.
 18. A method for hydrolyzing a lipid present in a soil and/or astain on a surface, comprising contacting the soil and/or the stain withthe polypeptide of any of claims 1-3 or the composition of any of claims4-15.
 19. An isolated polynucleotide encoding the polypeptide of any ofclaims 1-3.
 20. A nucleic acid construct or expression vector comprisingthe polynucleotide of claim 19 operably linked to one or more controlsequences that direct the production of the polypeptide in an expressionhost.
 21. A recombinant host cell comprising the polynucleotide of claim19 operably linked to one or more control sequences that direct theproduction of the polypeptide.
 22. A method of producing the polypeptideof any of claims 1-3, comprising: (a) cultivating a cell, which in itswild-type form produces the polypeptide, under conditions conducive forproduction of the polypeptide; and (b) recovering the polypeptide.
 23. Amethod of producing a polypeptide having lipase activity, comprising:(a) cultivating the host cell of claim 21 under conditions conducive forproduction of the polypeptide; and (b) recovering the polypeptide.